Stacia A. Sower

Sequencing of the sea lamprey (Petromyzon marinus) genome provides insights into vertebrate evolution.

Lampreys are representatives of an ancient vertebrate lineage that diverged from our own ∼500 million years ago. By virtue of this deeply shared ancestry, the sea lamprey (P. marinus) genome is uniquely poised to provide insight into the ancestry of vertebrate genomes and the underlying principles of vertebrate biology. Here, we present the first lamprey whole-genome sequence and assembly. We note challenges faced owing to its high content of repetitive elements and GC bases, as well as the absence of broad-scale sequence information from closely related species. Analyses of the assembly indicate that two whole-genome duplications likely occurred before the divergence of ancestral lamprey and gnathostome lineages. Moreover, the results help define key evolutionary events within vertebrate lineages, including the origin of myelin-associated proteins and the development of appendages. The lamprey genome provides an important resource for reconstructing vertebrate origins and the evolutionary events that have shaped the genomes of extant organisms.

The origins of the vertebrate hypothalamic–pituitary–gonadal (HPG) and hypothalamic–pituitary–thyroid (HPT) endocrine systems: New insights from lampreys

The acquisition of a hypothalamic-pituitary axis was a seminal event in vertebrate evolution leading to the neuroendocrine control of many complex functions including growth, reproduction, osmoregulation, stress and metabolism. Lampreys as basal vertebrates are the earliest evolved vertebrates for which there are demonstrated functional roles for two gonadotropin-releasing hormones (GnRHs) that act via the hypothalamic-pituitary-gonadal axis controlling reproductive processes. With the availability of the lamprey genome, we have identified a novel GnRH form (lamprey GnRH-II) and a novel glycoprotein hormone receptor, lGpH-R II (thyroid-stimulating hormone-like receptor). Based on functional studies, in situ hybridization and phylogenetic analysis, we hypothesize that the newly identified lamprey GnRH-II is an ancestral GnRH to the vertebrate GnRHs. This finding opens a new understanding of the GnRH family and can help to delineate the evolution of the complex neuro/endocrine axis of reproduction. A second glycoprotein hormone receptor (lGpH-R II) was also identified in the sea lamprey. The existing data suggest the existence of a primitive, overlapping yet functional HPG and HPT endocrine systems in this organism, involving one possibly two pituitary glycoprotein hormones and two glycoprotein hormone receptors as opposed to three or four glycoprotein hormones interacting specifically with three receptors in gnathostomes. We hypothesize that the glycoprotein hormone/glycoprotein hormone receptor systems emerged as a link between the neuro-hormonal and peripheral control levels during the early stages of gnathostome divergence. The significance of the results obtained by analysis of the HPG/T axes in sea lamprey may transcend the limited scope of the corresponding physiological compartments by providing important clues in respect to the interplay between genome-wide events (duplications), coding sequence (mutation) and expression control level evolutionary mechanisms in definition of the chemical control pathways in vertebrates.

Origins of Gonadotropin-Releasing Hormone (GnRH) in Vertebrates: Identification of a Novel GnRH in a Basal Vertebrate, the Sea Lamprey

We cloned a cDNA encoding a novel (GnRH), named lamprey GnRH-II, from the sea lamprey, a basal vertebrate. The deduced amino acid sequence of the newly identified lamprey GnRH-II is QHWSHGWFPG. The architecture of the precursor is similar to that reported for other GnRH precursors consisting of a signal peptide, decapeptide, a downstream processing site, and a GnRH-associated peptide; however, the gene for lamprey GnRH-II does not have introns in comparison with the gene organization for all other vertebrate GnRHs. Lamprey GnRH-II precursor transcript was widely expressed in a variety of tissues. In situ hybridization of the brain showed expression and localization of the transcript in the hypothalamus, medulla, and olfactory regions, whereas immunohistochemistry using a specific antiserum showed only GnRH-II cell bodies and processes in the preoptic nucleus/hypothalamus areas. Lamprey GnRH-II was shown to stimulate the hypothalamic-pituitary axis using in vivo and in vitro studies. Lamprey GnRH-II was also shown to activate the inositol phosphate signaling system in COS-7 cells transiently transfected with the lamprey GnRH receptor. These studies provide evidence for a novel lamprey GnRH that has a role as a third hypothalamic GnRH. In summary, the newly discovered lamprey GnRH-II offers a new paradigm of the origin of the vertebrate GnRH family. We hypothesize that due to a genome/gene duplication event, an ancestral gene gave rise to two lineages of GnRHs: the gnathostome GnRH and lamprey GnRH-II.

Revisiting the evolution of gonadotropin-releasing hormones and their receptors in vertebrates: secrets hidden in genomes.

Gonadotropin-releasing hormone (GnRH) and its G protein-coupled receptor, GnRHR, play a pivotal role in the control of reproduction in vertebrates. To date, many GnRH and GnRHR genes have been identified in a large variety of vertebrate species using conventional biochemical and molecular biological tools in combination with bioinformatic tools. Phylogenetic approaches, primarily based on amino acid sequence identity, make it possible to classify these multiple GnRHs and GnRHRs into several lineages. Four vertebrate GnRH lineages GnRH1, GnRH2, GnRH3, and GnRH4 (for lamprey) are well established. Four vertebrate GnRHR lineages have also been proposed-three for nonmammalian GnRHRs and mammalian GnRHR2 as well as one for mammalian GnRHR1. However, these phylogenetic analyses cannot fully explain the evolutionary origins of each lineage and the relationships among the lineages. Rapid and vast accumulation of genome sequence information for many vertebrate species, together with advances in bioinformatic tools, has allowed large-scale genome comparison to explore the origin and relationship of gene families of interest. The present review discusses the evolutionary mechanism of vertebrate GnRHs and GnRHRs based on extensive genome comparison. In this article, we focus only on vertebrate genomes because of the difficulty in comparing invertebrate and vertebrate genomes due to their marked divergence.

Evolutionary origin and divergence of PQRFamide peptides and LPXRFamide peptides in the RFamide peptide family: Insights from novel lamprey RFamide peptides

Among the RFamide peptide groups, PQRFamide peptides, such as neuropeptide FF (NPFF) and neuropeptide AF (NPAF), share a common C‐terminal Pro‐Gln‐Arg‐Phe‐NH2 motif. LPXRFamide (X = L or Q) peptides, such as gonadotropin‐inhibitory hormone (GnIH), frog growth hormone‐releasing peptide (fGRP), goldfish LPXRFamide peptide and mammalian RFamide‐related peptides (RFRPs), also share a C‐terminal Leu‐Pro‐Leu/Gln‐Arg‐Phe‐NH2 motif. Such a similar C‐terminal structure suggests that these two groups may have diverged from a common ancestral gene. In this study, we sought to clarify the evolutionary origin and divergence of these two groups, by identifying novel RFamide peptides from the brain of sea lamprey, one of only two extant groups of the oldest lineage of vertebrates, Agnatha. A novel lamprey RFamide peptide was identified by immunoaffinity purification using the antiserum against LPXRFamide peptide. The lamprey RFamide peptide did not contain a C‐terminal LPXRFamide motif, but had the sequence SWGAPAEKFWMRAMPQRFamide (lamprey PQRFa). A cDNA of the precursor encoded one lamprey PQRFa and two related peptides. These related peptides, which also had the C‐terminal PQRFamide motif, were further identified as mature endogenous ligands. Phylogenetic analysis revealed that lamprey PQRFamide peptide precursor belongs to the PQRFamide peptide group. In situ hybridization demonstrated that lamprey PQRFamide peptide mRNA is expressed in the regions predicted to be involved in neuroendocrine and behavioral functions. This is the first demonstration of the presence of RFamide peptides in the agnathan brain. Lamprey PQRFamide peptides are considered to have retained the most ancestral features of PQRFamide peptides.

Changes in plasma steroid and thyroid hormones and insulin during final maturation and spawning of the sea lamprey, Petromyzon marinus

Circulating levels of plasma estradiol-17 beta, androgens, thyroxine, triiodothyronine, immunoreactive insulin, plasma fatty acids, and protein were measured at interval during the period of final gonadal maturation, prior to spawning of male and female sea lampreys. Plasma estradiol levels fluctuated significantly and generally covaried in males and females through time. In females, possibly in relation to environmental changes, mean plasma estradiol levels peaked four times during the final spawning period but decreased sharply at the time of ovulation. In males, mean plasma estradiol peaked seven times during the final prespawning period and, in contrast to females, peaked significantly at the time of final spermiation. Plasma androgens were extremely low and covaried in males and females through time. Like plasma steroid profiles, there were coordinated changes in plasma triiodothyronine and thyroxine levels in males and females through the prespawning season. There was a slight increase in plasma insulin during the terminal maturation of the lampreys. However, at ovulation, the insulin levels abruptly decreased in females, whereas in males they remained unchanged. Plasma protein and fatty acid levels gradually decreased until ovulation/spermiation. At ovulation plasma fatty acid levels increased.

Similarities, differences, and unknowns in biology and management of three Parasitic lampreys of North America.

Abstract Sea lampreys, Petromyzon marinus, are invasive to the Laurentian Great Lakes where they have decimated native fishes. Great Lakes sea lampreys have been subjected to control measures for several decades, and the drive to control them has led to major advances in understanding their biology and in informing management. In contrast, anadromous sea and Pacific (Entosphenus tridentatus) lampreys have co-evolved with their oceanic prey. Both of these anadromous lampreys are in decline, and a limited amount of information on their biology has stymied conservation. The tendency has been to make biological inferences about anadromous lampreys based on the Great Lakes sea lamprey without justifiable evidence. We identify areas in which key information is missing for the juvenile (parasitic feeding) phase and adult freshwater spawning migrations, and compare and contrast information for these lampreys. Our comparisons reveal major differences, some intriguing similarities, and key unknowns that will requir...

Control of Gonadotropin Secretion by Follicle-Stimulating Hormone-Releasing Factor, Luteinizing Hormone-Releasing Hormone, and Leptin

Fractionation of hypothalamic extracts on a Sephadex G-25 column separates follicle-stimulating hormone-releasing factor (FSHRF) from luteinizing hormone-releasing hormone (LHRH). The FSH-releasing peak contained immunoreactive lamprey gonadotropin-releasing hormone (lGnRH) by radioimmunoassay, and its activity was inactivated by an antiserum specific to lGnRH. The identity of lGnRH-III with FSHRF is supported by studies with over 40 GnRH analogs that revealed that this is the sole analog with preferential FSH-releasing activity. Selective activity appears to require amino acids 5-8 of lGnRH-III. Chicken GnRH-II has slight selective FSH-releasing activity. Using a specific lGnRH-III antiserum, a population of lGnRH-III neurons was visualized in the dorsal and ventral preoptic area with axons projecting to the median eminence in areas shown previously to control FSH secretion based on lesion and stimulation studies. Some lGnRH-III neurons contained only this peptide, others also contained LHRH, and still others contained only LHRH. The differential pulsatile release of FSH and LH and their differential secretion at different times of the estrous cycle may be caused by differential secretion of FSHRF and LHRH. Both FSH and LHRH act by nitric oxide (NO) that generates cyclic guanosine monophosphate. lGnRH-III has very low affinity to the LHRH receptor. Biotinylated lGnRH-III (10(-9) M) labels 80% of FSH gonadotropes and is not displaced by LHRH, providing evidence for the existence of an FSHRF receptor. Leptin has equal potency as LHRH to release gonadotropins by NO. lGnRH-III specifically releases FSH, not only in rats but also in cows.

Neuronal systems immunoreactive with antiserum to lamprey gonadotropin-releasing hormone in the brain of Petromyzon marinus.

SummaryThe role of gonadotropin-releasing hormone (GnRH) in mammalian reproduction has been studied extensively; however, the role of a structurally different, but related, decapeptide is not well characterized in the most primitive class of vertebrates, Agnatha. Utilizing an antiserum directed to the recently characterized lamprey GnRH, we examined immunoreactive neuronal perikarya and nerve fibers in sections from the brain of the sea lamprey, Petromyzon marinus, using the unlabeled peroxidase-antiperoxidase method. Neuronal perikarya and fibers were immunopositive with antisera generated to lamprey GnRH and also to certain antisera generated to mammalian GnRH. Immunopositive neuronal perikarya were detected in an arcshaped population extending from ventral to dorsal preoptic areas. Fibers from these cells projected to the neurohypophysis via the preoptico-hypophyseal tract, but in addition also protruded into the third ventricle. Additionally, some fibers coursed along the external surface of the brain, and may also release GnRH into meningeal compartments. The presence of fully processed, mature decapeptide is indicated within neuronal perikarya, as well as in projecting nerve fibers and terminals. No reaction product was detected in sections incubated with an antiserum to the interior amino acid sequences of mammalian LHRH. This finding supports the structure reported for lamprey GnRH by Sherwood et al. (1986).

Relationship between brain gonadotropin-releasing hormone and final reproductive period of the adult male sea lamprey, Petromyzon marinus.

Gonadotropin-releasing hormone (GnRH) concentrations were measured in brains of adult male sea lamprey, Petromyzon marinus, during their final reproductive period. The lampreys were collected during their upstream migration in coastal New Hampshire rivers and sampled at the trap (referred to as Group A) or they were transferred to an artificial spawning channel (referred to as Group B). Plasma estradiol and progesterone were also measured, and histological examination of the gonadal stages was done as well. The concentrations of brain GnRH and plasma estradiol fluctuated significantly through time. There was a rise in brain concentrations of GnRH coincident with an increase in temperature just prior to spawning. In addition, there was a significant progressive correlation between increasing plasma estradiol and temperature in lampreys from Group B during the period studied. These studies provide evidence for progressive seasonal relationships between changes in brain GnRH and gametogenic and steroidogenic activity of the gonads in adult male sea lampreys during their final reproductive period.