Evolution of the endothelin pathway drove neural crest cell diversification.

Neural crest cells (NCCs) are migratory, multipotent embryonic cells that are unique to vertebrates and form an array of clade-defining adult features. The evolution of NCCs has been linked to various genomic events, including the evolution of new gene-regulatory networks1,2, the de novo evolution of genes3 and the proliferation of paralogous genes during genome-wide duplication events4. However, conclusive functional evidence linking new and/or duplicated genes to NCC evolution is lacking. Endothelin ligands (Edns) and endothelin receptors (Ednrs) are unique to vertebrates3,5,6, and regulate multiple aspects of NCC development in jawed vertebrates7-10. Here, to test whether the evolution of Edn signalling was a driver of NCC evolution, we used CRISPR-Cas9 mutagenesis11 to disrupt edn, ednr and dlx genes in the sea lamprey, Petromyzon marinus. Lampreys are jawless fishes that last shared a common ancestor with modern jawed vertebrates around 500 million years ago12. Thus, comparisons between lampreys and gnathostomes can identify deeply conserved and evolutionarily flexible features of vertebrate development. Using the frog Xenopus laevis to expand gnathostome phylogenetic representation and facilitate side-by-side analyses, we identify ancient and lineage-specific roles for Edn signalling. These findings suggest that Edn signalling was activated in NCCs before duplication of the vertebrate genome. Then, after one or more genome-wide duplications in the vertebrate stem, paralogous Edn pathways functionally diverged, resulting in NCC subpopulations with different Edn signalling requirements. We posit that this new developmental modularity facilitated the independent evolution of NCC derivatives in stem vertebrates. Consistent with this, differences in Edn pathway targets are associated with differences in the oropharyngeal skeleton and autonomic nervous system of lampreys and modern gnathostomes. In summary, our work provides functional genetic evidence linking the origin and duplication of new vertebrate genes with the stepwise evolution of a defining vertebrate novelty.

Discovery of prolactin-like in lamprey: Role in osmoregulation and new insight into the evolution of the growth hormone/prolactin family.

We used a representative of one of the oldest extant vertebrate lineages (jawless fish or agnathans) to investigate the early evolution and function of the growth hormone (GH)/prolactin (PRL) family. We identified a second member of the GH/PRL family in an agnathan, the sea lamprey (Petromyzon marinus). Structural, phylogenetic, and synteny analyses supported the identification of this hormone as prolactin-like (PRL-L), which has led to added insight into the evolution of the GH/PRL family. At least two ancestral genes were present in early vertebrates, which gave rise to distinct GH and PRL-L genes in lamprey. A series of gene duplications, gene losses, and chromosomal rearrangements account for the diversity of GH/PRL-family members in jawed vertebrates. Lamprey PRL-L is produced in the proximal pars distalis of the pituitary and is preferentially bound by the lamprey PRL receptor, whereas lamprey GH is preferentially bound by the lamprey GH receptor. Pituitary PRL-L messenger RNA (mRNA) levels were low in larvae, then increased significantly in mid-metamorphic transformers (stage 3); thereafter, levels subsided in final-stage transformers and metamorphosed juveniles. The abundance of PRL-L mRNA and immunoreactive protein increased in the pituitary of juveniles under hypoosmotic conditions, and treatment with PRL-L blocked seawater-associated inhibition of freshwater ion transporters. These findings clarify the origin and divergence of GH/PRL family genes in early vertebrates and reveal a function of PRL-L in osmoregulation of sea lamprey, comparable to a role of PRLs that is conserved in jawed vertebrates.

A pheromone antagonist liberates female sea lamprey from a sensory trap to enable reliable communication.

The evolution of male signals and female preferences remains a central question in the study of animal communication. The sensory trap model suggests males evolve signals that mimic cues used in nonsexual contexts and thus manipulate female behavior to generate mating opportunities. Much evidence supports the sensory trap model, but how females glean reliable information from both mimetic signals and their model cues remains unknown. We discovered a mechanism whereby a manipulative male signal guides reliable communication in sea lamprey (Petromyzon marinus). Migratory sea lamprey follow a larval cue into spawning streams; once sexually mature, males release a pheromone that mimics the larval cue and attracts females. Females conceivably benefit from the mimetic pheromone during mate search but must discriminate against the model cue to avoid orienting toward larvae in nearby nursery habitats. We tested the hypothesis that spawning females respond to petromyzonol sulfate (PZS) as a behavioral antagonist to avoid attraction to the larval cue while tracking the male pheromone despite each containing attractive 3-keto petromyzonol sulfate (3kPZS). We found 1) PZS inhibited electrophysiological responses to 3kPZS and abated preferences for 3kPZS when mixed at the same or greater concentrations, 2) larvae released more PZS than 3kPZS whereas males released more 3kPZS than PZS, and 3) mixtures of 3kPZS and PZS applied at ratios measured in larval and male odorants resulted in the discrimination observed between the natural odors. Our study elucidates how communication systems that arise via deception can facilitate reliable communication.

Upstream migrant sea lamprey (Petromyzon marinus) show signs of increasing oxidative stress but maintain aerobic capacity with age.

Following the parasitic juvenile phase of their life cycle, sea lamprey (Petromyzon marinus) mature into a reproductive but rapidly aging and deteriorating adult, and typically die shortly after spawning in May or June. However, pre-spawning upstream migrant sea lamprey can be maintained for several months beyond their natural lifespan when held in cold water (∼4-8 °C) under laboratory conditions. We exploited this feature to investigate the interactions between senescence, oxidative stress, and metabolic function in this phylogenetically ancient fish. We investigated how life history traits and mitochondria condition, as indicated by markers of oxidative stress (catalase activity, lipid peroxidation) and aerobic capacity (citrate synthase activity), changed in adult sea lamprey from June to December after capture during their upstream spawning migration. Body mass but not liver mass declined with age, resulting in an increase in hepatosomatic index. Both effects were most pronounced in males, which also tended to have larger livers than females. Lamprey experienced greater oxidative stress with age, as reflected by increasing activity of the antioxidant enzyme catalase and increasing levels of lipid peroxidation in liver mitochondrial isolates over time. Surprisingly, the activity of citrate synthase also increased with age in both sexes. These observations implicate mitochondrial dysfunction and oxidative stress in the senescence of sea lamprey. Due to their unique evolutionary position and the technical advantage of easily delaying the onset of senescence in lampreys using cold water, these animals could represent an evolutionary unique and tractable model to investigate senescence in vertebrates.

Plasma metabolomic profiles reveal sex- and maturation-dependent metabolic strategies in sea lamprey (Petromyzon marinus).

INTRODUCTION: Adult sea lamprey (Petromyzon marinus) cease feeding and migrate to spawning streams where males build nests, undergo final sexual maturation, and subsequently produce and release large quantities of bile acid pheromones that attract mature females. These animals are predicted to rearrange their metabolic pathways drastically to support their reproductive strategies, presenting advantageous opportunities to examine how sex and the maturation processes affect metabolism. OBJECTIVES: The objective is to investigate the metabolic differences between sexes and maturation states in sea lamprey that support changes in physiological functions. METHODS: We compared plasma metabolomes of spawning and prespawning sea lamprey in both sexes using both non-targeted and targeted metabolomics approaches using UPLC/MS-MS with electrospray ionization in both positive and negative modes. The data were processed using Progenesis QI, Compound Discoverer and XCMS softwares for alignment, peak picking, and deconvolution of the peaks. Principle component analyses (PCA) and partial least squares discriminant analyses (PLS-DA) were performed using SIMCA and Metaboanalyst softwares to identify discriminating features, followed by fragmentation matching with extensive database search and pathway mapping. RESULTS: The pheromonal bile acid biosynthesis was upregulated significantly in males compared to females. Spermiating males further upregulated bile acid biosynthesis by altering amino acid metabolisms, upregulating cofactors and nucleotide metabolisms, but downregulating carbohydrate and energy metabolisms. CONCLUSION: Plasma metabolomes are sex- and maturation-dependent and reflect the special metabolic demands at each life stage and reproductive strategy.

Spermine in semen of male sea lamprey acts as a sex pheromone.

Semen is fundamental for sexual reproduction. The non-sperm part of ejaculated semen, or seminal plasma, facilitates the delivery of sperm to the eggs. The seminal plasma of some species with internal fertilization contains anti-aphrodisiac molecules that deter promiscuity in post-copulatory females, conferring fitness benefits to the ejaculating male. By contrast, in some taxa with external fertilization such as fish, exposure to semen promotes spawning behaviors. However, no specific compounds in semen have been identified as aphrodisiac pheromones. We sought to identify a pheromone from the milt (fish semen) of sea lamprey (Petromyzon marinus), a jawless fish that spawns in lek-like aggregations in which each spermiating male defends a nest, and ovulatory females move from nest to nest to mate. We postulated that milt compounds signal to ovulatory females the presence of spawning spermiating males. We determined that spermine, an odorous polyamine initially identified from human semen, is indeed a milt pheromone. At concentrations as low as 10-14 molar, spermine stimulated the lamprey olfactory system and attracted ovulatory females but did not attract males or pre-ovulatory females. We found spermine activated a trace amine-associated receptor (TAAR)-like receptor in the lamprey olfactory epithelium. A novel antagonist to that receptor nullified the attraction of ovulatory females to spermine. Our results elucidate a mechanism whereby a seminal plasma pheromone attracts ready-to-mate females and implicates a possible conservation of the olfactory detection of semen from jawless vertebrates to humans. Milt pheromones may also have management implications for sea lamprey populations.

Variation in the Transcriptome Response and Detoxification Gene Diversity Drives Pesticide Tolerance in Fishes.

Pesticides are critical for invasive species management but often have negative effects on nontarget native biota. Tolerance to pesticides should have an evolutionary basis, but this is poorly understood. Invasive sea lamprey (Petromyzon marinus) populations in North America have been controlled with a pesticide lethal to them at lower concentrations than native fishes. We addressed how interspecific variation in gene expression and detoxification gene diversity confer differential pesticide sensitivity in two fish species. We exposed sea lamprey and bluegill (Lepomis macrochirus), a tolerant native species, to 3-trifluoromethyl-4-nitrophenol (TFM), a pesticide commonly used in sea lamprey control. We then used whole-transcriptome sequencing of gill and liver to characterize the cellular response in both species. Comparatively, bluegill exhibited a larger number of detoxification genes expressed and a larger number of responsive transcripts overall, which likely contributes to greater tolerance to TFM. Understanding the genetic and physiological basis for pesticide tolerance is crucial for managing invasive species.

Granulin as an important immune molecule involved in lamprey tissue repair and regeneration by promoting cell proliferation and migration.

Progranulin (PGRN) is an autocrine growth factor that regulates cell proliferation, migration, wound healing, and tissue repair in mammals. Lamprey is the most primitive of the extant vertebrates and is regarded as the survivor of a once flourishing group of paleozoic vertebrates, with a history of more than 500 million years. To date, the evolutionary dynamics and the underlying function of the PGRNs remain largely unclear in lamprey. Here, we screened four genes encoding PGRNs from the genomes of Lethenteron reissneri and Petromyzon marinus, including one long form (named Lr-PGRN-L) and three short forms (named Lr-PGRN-S1, Lr-PGRN-S2, and Lr-PGRN-S3), and performed phylogenetic tree, functional domain, and synteny analyses to identify the evolutionary history of the four Lr-PGRNs. In addition, the expressions of the four Lr-pgrn family genes and the immune response against various pathogenic challenges were also investigated. We found that these genes were widely distributed in various tissues of lamprey and performed a variety of functions. Moreover, our results suggest that Lr-PGRN-S1 induces cell migration and proliferation, and is involved in repair after skin and spinal cord injury under appropriate conditions. Our findings are valuable because they improve the understanding of the evolutionary relationship of vertebrate pgrn genes, as well as providing new insights into the diverse and important roles of Lr-PGRNs.

Prediction of ATPase cation transporting 13A2 molecule in Petromyzon marinus and pan-cancer analysis into human tumors from an evolutionary perspective.

The ATPase cation transporting 13A2 protein (ATP13A2), which maintains the homeostasis of mitochondria and lysosomes, plays a significant role in human neurodegenerative diseases and cancer. Through constructing a lamprey proteome database, employing multiple sequence alignment and phylogenetic analysis, 5 ATP13A2 proteins from Petromyzon marinus (Pm-ATP13A2) were identified based on the evolutionary perspective. The motif and domain analysis showed that the ATP13A2 protein was conserved. The multiple phosphorylation sites and transmembrane structures highlighted the characteristics of ATP13A2 as the P-ATPase-V cation transporting protein. Based on the information provided by the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, this study was conducted as a preliminary investigation of the carcinogenic effects of the ATP13A2 gene in a variety of tumors. The ATP13A2 was strongly expressed in most tumors, except in two types of nervous system tumors glioblastoma multiforme (GBM) and brain lower grade glioma (LGG). Moreover, the expression of ATP13A2 was strongly correlated with the prognosis of tumor patients. The high expression of ATP13A2 was obviously related to the poor prognosis of LGG. The poor prognosis of LGG patients may affect the ATP13A2 expression through the immune cells and radiotherapy. Also, cancer-related fibroblast infiltration was observed. All in all, this work offers more insights into the molecular evolution of the ATP13A2 protein and facilitates the understanding of the carcinogenic effects of the ATP13A2 in different tumors. Our discussion also promotes the study into the successful evolution of the vertebrate brain and the mechanism of clinical brain-related diseases.

Corticosteroid control of Na+/K+-ATPase in the intestine of the sea lamprey (Petromyzon marinus).

Anadromous sea lamprey (Petromyzon marinus) larvae undergo a months-long true metamorphosis during which they develop seawater (SW) tolerance prior to downstream migration and SW entry. We have previously shown that intestinal Na+/K+-ATPase (NKA) activity increases during metamorphosis and is critical to the osmoregulatory function of the intestine in SW. The present study investigated the role of 11-deoxycortisol (S) in controlling NKA in the anterior (AI) and posterior (PI) intestine during sea lamprey metamorphosis. In a tissue profile, nka mRNA and protein were most abundant in the gill, kidney, and AI. During metamorphosis, AI nka mRNA increased 10-fold, whereas PI nka mRNA did not change. Specific corticosteroid receptors were found in the AI, which had a higher binding affinity for S compared to 11-deoxycorticosterone (DOC). In vivo administration of S in mid-metamorphic lamprey upregulated NKA activity 3-fold in the AI and PI, whereas administration of DOC did not affect intestinal NKA activity. During a 24 h SW challenge test, dehydration of white muscle moisture was rescued by prior treatment with S, which was associated with increased intestinal nka mRNA and NKA activity. These results indicate that intestinal osmoregulation in sea lamprey is a target for control by S during metamorphosis and the development of SW tolerance.

Osmoregulatory role of the intestine in the sea lamprey (Petromyzon marinus).

Lampreys are the most basal vertebrates with an osmoregulatory strategy. Previous research has established that the salinity tolerance of sea lamprey increases dramatically during metamorphosis, but underlying changes in the gut have not been examined. In the present work, we examined changes in intestinal function during metamorphosis and seawater exposure of sea lamprey (Petromyzon marinus). Fully metamorphosed juvenile sea lamprey had 100% survival after direct exposure to 35 parts per thousand seawater (SW) and only slight elevations in plasma chloride (Cl-) levels. Drinking rates of sea lamprey juveniles in seawater were 26-fold higher than juveniles in freshwater (FW). Na+-K+-ATPase (NKA) activity in the anterior and posterior intestine increased 12- and 3-fold, respectively, during metamorphosis, whereas esophageal NKA activity was lower than in the intestine and did not change with development. Acclimation to SW significantly enhanced NKA activity in the posterior intestine but did not significantly change NKA activity in the anterior intestine, which remained higher than that in the posterior intestine. Intestinal Cl- and water uptake, which were observed in ex vivo preparations of anterior and posterior intestine under both symmetric and asymmetric conditions, were higher in juveniles than in larvae and were similar in magnitude of those of teleost fish. Inhibition of NKA by ouabain in ex vivo preparations inhibited intestinal water absorption by 64%. Our results indicate drinking and intestinal ion and water absorption are important to osmoregulation in SW and that preparatory increases in intestinal NKA activity are important to the development of salinity tolerance that occurs during sea lamprey metamorphosis.

Sex-dependent pheromonal effects on steroid hormone levels in sea lampreys (Petromyzon marinus).

Sea lampreys (Petromyzon marinus) are basal vertebrates that exhibit reproductive control via a hypothalamic-pituitary-gonadal axis. The function and evolution of the hypothalamic and pituitary peptide hormones are well studied in this species, whereas the functions of classical sex steroid hormones have not been well established due to their low or non-detectable plasma levels. Sea lamprey pheromone 3-keto petromyzonol sulfate (3kPZS) has been shown to increase while 3-keto allocholic acid (3kACA) decreases plasma 15α-hydroxyprogesterone (15αP) levels in prespermiating males (PSM) but not in preovulatory females (POF). However, spermiating male washings that contain both 3kPZS and 3kACA facilitate spawning in both sexes. Therefore, we wondered if the effects of pheromones on POF were elicited by classical steroid hormones such as progesterone, androstenedione, testosterone and estradiol. We hypothesized that waterborne 3kACA and 3kPZS differentially alter steroid hormone levels in prespawning sea lampreys. We determined the sex differences and pheromonal effects on steroid hormone levels in prespawning sea lampreys using sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) methods. Some PSM became spermiating (SM) at the time of sample collection, and those data were treated as a separate group. We found that males contained more androstenedione and progesterone in the gonad and more estradiol and testosterone in the plasma than POF, whereas POF contained more gonadal testosterone than males (no detectable gonadal testosterone). In POF, 3kPZS decreased gonadal androstenedione but increased gonadal progesterone and plasma estradiol, whereas 3kACA only increased gonadal progesterone levels. Exposure to 3kPZS for 4 h increased plasma 15αP in POF and SM, and gonadal 15αP in POF. Interestingly, 3kACA or 3kPZS depleted gonadal or plasma 15αP in PSM at various time points. On the other hand, both pheromones had no significant effect on androstenedione, progesterone or estradiol levels in males. Plasma testosterone levels did not change after pheromone exposure in both sexes. We conclude that sea lamprey pheromones 3kACA and 3kPZS induced differential steroidal responses in POF, PSM and SM.

Waterborne pheromones modulate gonadotropin-inhibitory hormone levels in sea lamprey (Petromyzon marinus).

The relationships between pheromone stimuli and neuropeptides are not well established in vertebrates due to the limited number of unequivocally identified pheromone molecules. The sea lamprey (Petromyzon marinus) is an advantageous vertebrate model to study the effects of pheromone exposure on neuropeptides since many pheromone molecules and neuropeptides have been identified in this species. Sexually mature male sea lamprey release pheromones 7α, 12α, 24-trihydroxy-5α-cholan-3-one 24-sulfate (3 keto-petromyzonol sulfate, 3kPZS) and 7α, 12α-dihydroxy-5α-cholan-3-one-24-oic acid (3-keto allocholic acid, 3kACA) that differentially regulate gonadotropin-releasing hormone (lGnRH) and steroid levels in sexually immature sea lamprey. However, the effects of these pheromones on gonadotropin-inhibitory hormones (GnIHs), hypothalamic neuropeptides that regulate lGnRH release, are still elusive. In this report, we sought to examine the effects of waterborne pheromones on lamprey GnIH-related neuropeptide levels in sexually immature sea lamprey. Ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analyses revealed sex differences in GnIH-related neuropeptide levels in the brain and plasma of immature sea lamprey. Exposure to 3kPZS and 3kACA exerted differential effects on GnIH-related neuropeptide levels in both sexes, but the effects were more prominent in female brains. We conclude that sea lamprey pheromones regulate GnIH-related neuropeptide levels in a sexually dimorphic manner.

Muscle fatty acid profiles of sea lamprey (Petromyzon marinus L.) indicate the use of fast metabolized energy during ontogenesis.

A comprehensive characterization of muscle's FA composition of sea lamprey ammocoetes and adults was performed to test the hypothesis that larvae, and early spawning migrants have a similar FA profile prior to metamorphosis and to spawning migration. Subsequently, the role played by FA signature in these two highly demanding stages of life cycle was inferred. The results confirm that muscle represents an important fat reservoir, and the FA trophic markers revealed the importance of bacteria as sources of iso and anteiso FA and the strong trophic representation of benthic phytoplankton (diatoms) to larvae muscle FA profile. In early spawning migrants, the significance of marine food web to FA muscle profile is highlighted by the presence of FA signatures characteristics of herbivorous calanoid copepods. Although both life cycle phases studied do not share the same muscle FA signature, there is a part of the profile that is common, which is characterized by FA used in ß-oxidation, such as C18:1ω9 but also by medium chain FA and PUFA which points that PUFA are spared as fuel to ß-oxidation process and probably used to the development of tissues membranes (ammocoetes) and gonadal development and eicosanoid production among others (early spawning migrants). Further studies on FA profile are necessary to elucidate the FA role either during different life stages (ontogeny) or in the distinct habitats frequented (freshwater versus marine) by this diadromous species.

Metabolism of a sea lamprey pesticide by fish liver enzymes part A: identification and synthesis of TFM metabolites.

The sea lamprey (Petromyzon marinus) is a destructive invasive species in the Great Lakes that contributed to the collapse of native fish populations in the mid-1900s. 3-Trifluoromethyl-4-nitrophenol (TFM) is a selective pesticide that has been applied to sea lamprey infested tributaries of the Great Lakes to kill larvae since the 1960s and has reduced the populations by as much as 90%. However, the metabolism of TFM by sea lamprey and non-target species is not fully illuminated. Elucidation of TFM metabolism is critical for understanding its mode of action and possible environmental impact. Here, we describe the screening, identification, synthesis and structural characterization of TFM metabolites in livers from sea lamprey and three non-target species that differ in their ability to survive TFM exposure. We identified glucuronidation, sulfation, N-acetylation, glutathione conjugation, and aromatic nitro group reduction as potential detoxification mechanisms. Seven metabolites were synthesized for use as markers of TFM metabolism in fish. Quantitative 1H NMR was used to assay synthesized metabolite stock solutions that were then used as standard material to develop a quantitative LC-MS/MS method for TFM metabolites.

Metabolism of a sea lamprey pesticide by fish liver enzymes part B: method development and application in quantification of TFM metabolites formed in vivo.

The sea lamprey (Petromyzon marinus) is a destructive invasive species in the Great Lakes. Since the 1960s, tons of the lampricide 3-trifluoromethyl-4-nitrophenol (TFM) has been applied to selected tributaries each year to eliminate or reduce sea lamprey larval populations. Therefore, the environmental impact of TFM needs to be evaluated. However, the metabolism of TFM and its mechanism of selective toxicity in sea lamprey is not yet fully understood. Based upon our previous report on the identification, synthesis, and characterization of TFM metabolites observed in liver incubates from sea lamprey and non-target fishes, we now provide a robust assay for quantifying TFM and its metabolites in fish liver tissue. This method is important for assessing bioaccumulation of TFM in the ecosystems. The compounds purified in our previous report were used to develop and validate a quantitative ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for TFM and TFM metabolites formed in vivo. Several sample preparation techniques were compared, and a protein precipitation method was selected. The unavailability of stable isotopic internal standards was overcome by using a matrix matching method. After a thorough validation, this method was applied to determine the concentrations of TFM and its metabolites in fish liver tissues from animals exposed to TFM, and in the comparison between dead animals and survivors. Seven of eight expected metabolites were observed, some for the first time in vivo. Our results indicate that in vivo nitroreduction, glucuronidation, sulfation, and glutathione conjugation are involved in TFM metabolism in sea lamprey.

Co-localization of three gonadotropin-releasing hormone transcripts in larval, parasitic, and adult sea lamprey brains.

RNA expression of lamprey gonadotropin-releasing hormone (lGnRH)-I, -II, and -III was demonstrated in the brains of larval, parasitic phase and adult sea lampreys, Petromyzon marinus, using a highly sensitive triple-label in situ hybridization technique. In female larval lampreys, lGnRH-I and-II were co-expressed in the same neurons throughout the olfactory bulbs, preoptic area (POA), and rhombencephalon (hindbrain); lGnRH-I, -II and -III were triple co-expressed in the hypothalamus and in the paranuclear region of neuronal somas in the rhombencephalon. In female parasitic phase lampreys, lGnRH-I and -II were co-expressed in the POA, thalamus, and preoptico-neurohypophyseal tract (PNT); lGnRH-III was minimally triple co-expressed with lGnRH-I and -II in the hypothalamus. In adult female lampreys, lGnRH-I and -III were co-expressed in the hypothalamus; lGnRH-I was also expressed in the neurohypophysis (NH). In adult male lampreys, lGnRH-I and-III were co-expressed in the primordial hippocampus, POA, thalamus, hypothalamus, NH, and PNT; lGnRH-I was also expressed in the epithalamus. In summary, we provide the first study using in situ hybridization of all three lGnRHs (lGnRH-I, -II, and -III) at three major life stages (larval, parasitic, and adult) of lampreys, which strongly supports previous immunohistological studies and suggests that lGnRH-I and -II are the predominant lGnRHs in larval and parasitic phase lampreys, and that lGnRH-I and -III are the predominant lGnRHs in adult female and male lampreys. Therefore, our results show that lGnRH-I, -II, and -III have different localization and co-expression in the development and sexual maturation of lampreys, which may suggest unique physiological roles at each life stage and sex in the developing and mature lamprey brain.

Expression of two glycoprotein hormone receptors in larval, parasitic phase, and adult sea lampreys.

All jawed vertebrates have three canonical glycoprotein hormones (GpHs: luteinizing hormone, LH; follicle stimulating hormone, FSH; and thyroid stimulating hormone, TSH) with three corresponding GpH receptors (GpH-Rs: LH-R, FSH-R, and TSH-R). In contrast, we propose that the jawless vertebrate, the sea lamprey (Petromyzon marinus), only has two pituitary glycoprotein hormones, lamprey (l)GpH and l-thyrostimulin, and two functional glycoprotein receptors, lGpH-R I and II. It is not known at this time whether there is a specific receptor for lGpH and l-thyrostimulin, or if both GpHs can differentially activate the lGpH-Rs. In this report, we determined the RNA expression of lGpH-R I and II in the gonads and thyroids of larval, parasitic phase, and adult lampreys. A highly sensitive dual-label fluorescent in situ hybridization technique (RNAScope™) showed lGpH-R I expression in the ovaries of larval lamprey, and co-localization and co-expression of lGpH-R I and II in the ovaries of parasitic phase and adult lampreys. Both receptors were also highly co-localized and co-expressed in the endostyle of larval lamprey and thyroid follicles of parasitic and adult lampreys. In addition, we performed in vivo studies to determine the actions of lamprey gonadotropin releasing hormones (lGnRHs) on lGpH-R I and II expression by real time PCR, and determined plasma concentrations of estradiol and thyroxine. Administration of lGnRH-III significantly (p ≤ 0.01) increased lGpHR II expression in the thyroid follicles of adult female lampreys but did not cause a significant increase in RNA expression of lGpH-R I and II in ovaries. Concomitantly, there was a significant increase (p ≤ 0.01) of plasma estradiol without any significant changes of plasma thyroxine concentrations in response to treatment to lGnRH-I, -II, or -III. In summary, our results provide supporting evidence that the lamprey pituitary glycoprotein hormones may differentially activate the lamprey GpH-Rs in regulating both thyroid and gonadal activities during each of the three life stages of the sea lamprey.

Spatial and Ontogenetic Variation in Mercury in Lake Superior Basin Sea Lamprey (Petromyzon marinus).

Mercury concentrations were measured in eggs, larvae, and adult spawning-phase sea lampreys (Petromyzon marinus) collected in tributaries of Lake Superior to investigate spatial and ontogenetic variation. There were significant differences in mercury concentrations between all three life stages, with levels highest in adults (mean = 3.01 µg/g), followed by eggs (mean = 0.942 µg/g), and lowest in larvae (mean = 0.455 µg/g). There were no significant differences in mercury concentrations by location for any life stage or by sex in adults. Mercury was not correlated with adult or larval lamprey length or mass. Mercury levels in adult lampreys exceeded U.S. and Canadian federal guidelines for human consumption. Mercury concentrations in all life stages exceeded criteria for the protection of piscivorous wildlife, posing a threat to local fish, birds, and mammals. High mercury levels in adult lampreys combined with their semelparous life history make them a potential source of lake-derived mercury to spawning streams.

Comprehensive histological and immunological studies reveal a novel glycoprotein hormone and thyrostimulin expressing proto-glycotrope in the sea lamprey pituitary.

In the adenohypophysis (anterior pituitary) of all gnathostomes, there are six tropic cell types: corticotropes, melanotropes, somatotropes, lactotropes, gonadotropes and thyrotropes; each cell type produces specific tropic hormones. In contrast, we report in this study that there are only four tropic cell types in the sea lamprey (Petromyzon marinus) adenohypophysis. We specifically focused on the cell types that produce the glycoprotein hormones (GpHs). The gnathostome adenohypophyseal GpHs are follicle-stimulating hormone (FSH), luteinizing hormone (LH), thyroid-stimulating hormone (TSH), and thyrostimulin. However, lampreys only have two heterodimeric adenohypophyseal GpHs consisting of unique α and ß subunits, lamprey GpH (lGpH) (lGpA2/lGpHß) and thyrostimulin (lGpA2/lGpB5). We used an array of histological techniques to determine the (co)-localization and (co)-expression of the lGpH and thyrostimulin subunits in the lamprey adenohypophysis at different life stages (larval, parasitic, adult) and to identify their synthesizing cell(s). The thyrostimulin subunits (lGpA2/lGpB5) were co-expressed throughout the adenohypophysis (larval, parasitic, and adult), while the GpH ß-subunit (lGpHß) exhibited localized distribution (adult); all three subunits were co-localized and co-expressed, suggesting that both GpHs are synthesized in the same cells, novel proto-glycotropes, in specific adenohypophyseal regions at different life stages. In summary, we provide the first comprehensive study using histology, transmission electron microscopy, in situ hybridization and immunohistochemistry that strongly supports further evidence for four definitive adenohypophyseal cell types in the lamprey, including: corticotropes, somatotropes, melanotropes, and the first identification of a novel proto-glycotrope. In addition, our studies show that there is developmental and region-specific co-localization and co-expression of lGpH and thyrostimulin in the lamprey adenohypophysis.