Determination of thyroid hormones in lake sturgeon (Acipenser fulvescens) tissues at different developmental stages using a sensitive liquid chromatography-mass spectrometry method.

Thyroid hormones (TH) are known to play an important role in the growth and development of vertebrates. In fish species, TH regulates the larval-juvenile metamorphosis, and is crucial for development during early life stages. Monitoring the variations in TH levels at different life stages can provide insights into the regulation of metamorphosis and fish development. In this study, we developed an extremely sensitive method for the quantification of thyroxine (T4), triiodothyronine (T3), and reverse-triiodothyronine (rT3), in lake sturgeon (Acipenser fulvescens) tissues from eggs, free embryos, larvae, and juveniles. The target compounds were extracted by an enzymatic digestion method, followed by protein precipitation. Further cleanup was performed by liquid-liquid extraction (LLE) and solid phase extraction (SPE) using SampliQ OPT cartridges. The liquid-chromatography tandem mass spectrometry (LC-MS/MS) method used to quantify TH compounds showed remarkably high sensitivity with the limit of detection (LOD) and the limit of quantification (LOQ) ranging from < 1 pg/mL to 10 pg/mL and linearity in the range of 10-50,000 pg/mL. This method was validated for tissue samples across several early developmental stages and was checked for intra- and inter-day accuracy (78.3-111.2 %) and precision (0.1-4.9 %), matrix effect (75.4-134.1 %), and recovery (41.2-69.0 %). The method was successfully applied for the quantification and comparison of T4, T3 and rT3 in hatchery raised lake sturgeon samples collected at unique time points (i.e., days post fertilization dpf) including fertilized eggs (11 dpf), free embryos (14 dpf), larvae (22 dpf), juveniles (40 dpf) and older juveniles (74 dpf). With modifications, this method could be applied to other species important for agriculture or conservation.

Disruption of T-box transcription factor eomesa results in abnormal development of median fins in Oujiang color common carp Cyprinus carpio.

Median fins are thought to be ancestors of paired fins which in turn give rise to limbs in tetrapods. However, the developmental mechanisms of median fins remain largely unknown. Nonsense mutation of the T-box transcription factor eomesa in zebrafish results in a phenotype without dorsal fin. Compared to zebrafish, the common carp undergo an additional round of whole genome duplication, acquiring an extra copy of protein-coding genes. To verify the function of eomesa genes in common carp, we established a biallelic gene editing technology in this tetraploidy fish through simultaneous disruption of two homologous genes, eomesa1 and eomesa2. We targeted four sites located upstream or within the sequences encoding the T-box domain. Sanger sequencing data indicated the average knockout efficiency was around 40% at T1-T3 sites and 10% at T4 site in embryos at 24 hours post fertilization. The individual editing efficiency was high to about 80% at T1-T3 sites and low to 13.3% at T4 site in larvae at 7 days post fertilization. Among 145 mosaic F0 examined at four months old, three individuals (Mutant 1-3) showed varying degrees of maldevelopment in the dorsal fin and loss of anal fin. Genotyping showed the genomes of all three mutants were disrupted at T3 sites. The null mutation rates on the eomesa1 and eomesa2 loci were 0% and 60% in Mutant 1, 66.7% and 100% in Mutant 2, and 90% and 77.8% in Mutant 3, respectively. In conclusion, we demonstrated a role of eomesa in the formation and development of median fins in Oujiang color common carp and established an method that simultaneously disrupt two homologous genes with one gRNA, which would be useful in genome editing in other polyploidy fishes.

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.

Convergent olfactory trace amine-associated receptors detect biogenic polyamines with distinct motifs via a conserved binding site.

Biogenic amines activate G-protein-coupled receptors (GPCRs) in the central nervous system in vertebrate animals. Several biogenic amines, when excreted, stimulate trace amine-associated receptors (TAARs), a group of GPCRs in the main olfactory epithelium, and elicit innate behaviors. How TAARs recognize amines with varying numbers of amino groups is largely unknown. We reasoned that a comparison between lamprey and mammalian olfactory TAARs, which are thought to have evolved independently but show convergent responses to polyamines, may reveal structural determinants of amine recognition. Here, we demonstrate that sea lamprey TAAR365 (sTAAR365) responds strongly to biogenic polyamines cadaverine, putrescine, and spermine, and shares a similar response profile as a mammalian TAAR (mTAAR9). Docking and site-directed mutagenesis analyses show that both sTAAR365 and mTAAR9 recognize the two amino groups of cadaverine with the conserved Asp3.32 and Tyr6.51 residues. sTAAR365, which has remarkable sensitivity for cadaverine (EC50 = 4 nM), uses an extra residue, Thr7.42, to stabilize ligand binding. These cadaverine recognition sites also interact with amines with four and three amino groups (spermine and spermidine, respectively). Glu7.36 of sTAAR365 cooperates with Asp3.32 and Thr7.42 to recognize spermine, whereas mTAAR9 recognizes spermidine through an additional aromatic residue, Tyr7.43. These results suggest a conserved mechanism whereby independently evolved TAAR receptors recognize amines with two, three, or four amino groups using the same recognition sites, at which sTAAR365 and mTAAR9 evolved distinct motifs. These motifs interact directly with the amino groups of the polyamines, a class of potent and ecologically important odorants, mediating olfactory signaling.

Bile acid production is life-stage and sex-dependent and affected by primer pheromones in the sea lamprey.

Pheromonal bile salts are important for sea lampreys (Petromyzon marinus Linnaeus) to complete their life cycle. The synthesis and release of a releaser/primer pheromone 3-keto petromyzonol sulfate (3kPZS) by spermiating males have been well characterized. 3kPZS evokes sexual behaviors in ovulatory females, induces immediate 3kPZS release in spermiating males, and elicits neuroendocrine responses in prespawning adults. Another primer pheromone released by spermiating males, 3-keto allocholic acid (3kACA), antagonizes the neuroendocrine effects of 3kPZS in prespermiating males. However, the effects of 3kACA and 3kPZS on pheromone production in prespawning adults is unclear. To understand the foundation of pheromone production, we examined sea lamprey bile salt levels at different life stages. To investigate the priming effects of 3kACA and 3kPZS, we exposed prespawning adults with vehicle or synthetic 3kACA or 3kPZS. We hypothesized that endogenous bile salt levels were life-stage and sex-dependent, and differentially affected by 3kACA and 3kPZS in prespawning adults. Using ultra-performance liquid chromatography tandem mass spectrometry, we found that sea lampreys contained distinct mixtures of bile salts in the liver and plasma at different life stages. Males usually contained higher amounts of bile salts than females. Petromyzonamine disulfate was the most abundant C27 bile salt and petromyzonol sulfate was the most abundant C24 bile salt. Waterborne 3kACA and 3kPZS exerted differential effects on bile salt production in the liver and gill, their circulation and clearance in the plasma, and their release into water. We conclude that bile salt levels are life-stage and sex-dependent and differentially affected by primer pheromones.

Global tissue transcriptomic analysis to improve genome annotation and unravel skin pigmentation in goldfish.

Goldfish is an ornamental fish with diverse phenotypes. However, the limited genomic resources of goldfish hamper our understanding of the genetic basis for its phenotypic diversity. To provide enriched genomic resources and infer possible mechanisms underlying skin pigmentation, we performed a large-scale transcriptomic sequencing on 13 adult goldfish tissues, larvae at one- and three-days post hatch, and skin tissues with four different color pigmentation. A total of 25.52 Gb and 149.80 Gb clean data were obtained using the PacBio and Illumina platforms, respectively. Onto the goldfish reference genome, we mapped 137,674 non-redundant transcripts, of which 5.54% was known isoforms and 78.53% was novel isoforms of the reference genes, and the remaining 21,926 isoforms are novel isoforms of additional new genes. Both skin-specific and color-specific transcriptomic analyses showed that several significantly enriched genes were known to be involved in melanogenesis, tyrosine metabolism, PPAR signaling pathway, folate biosynthesis metabolism and so on. Thirteen differentially expressed genes across different color skins were associated with melanogenesis and pteridine synthesis including mitf, ednrb, mc1r, tyr, mlph and gch1, and xanthophore differentiation such as pax7, slc2a11 and slc2a15. These transcriptomic data revealed pathways involved in goldfish pigmentation and improved the gene annotation of the reference genome.

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.

TGF-ß Signaling Plays a Pivotal Role During Developmental Biliary Atresia in Sea Lamprey (Petromyzon marinus).

Biliary atresia (BA) is a rare neonatal disease with unknown causes. Approximately 10% of BA cases develop in utero with other congenital defects that span a large spectrum of disease variations, including degeneration of the gall bladder and bile duct as well as malformation of the liver, intestines, and kidneys. Similar developmental alterations are manifested in a unique animal model, the sea lamprey (Petromyzon marinus), in which BA occurs naturally during metamorphosis. With the likelihood of conserved developmental mechanisms underlying organogenesis and degeneration, lamprey developmental BA may be a useful model to infer mechanisms underlying human embryonic BA. We reasoned that hepatobiliary transcriptomes regulate the transition between landmark stages of BA. Therefore, we examined sea lamprey hepatobiliary transcriptomes at four stages (M0, metamorphic stage 0 or larval stage, no BA; M2, metamorphic stage 2, onset of BA; M5, metamorphic stage 5, BA, and heightened hepatocyte proliferation and reorganization; and JV, juvenile, completion of BA) using messenger RNA sequencing and Kyoto Encyclopedia of Genes and Genomes pathway analyses. We found gene-expression patterns associated with the transition between these stages. In particular, transforming growth factor ß (TGF-ß), hedgehog, phosphatidylinositol-4,5-bisphosphate 3-kinase-Akt, Wnt, and mitogen-activated protein kinase pathways were involved during biliary degeneration. Furthermore, disrupting the TGF-ß signaling pathway with antagonist or small interfering RNA treatments at the onset of BA delayed gall bladder and bile duct degeneration. Conclusion: Distinctive gene-expression patterns are associated with the degeneration of the biliary system during developmental BA. In addition, disrupting TGF-ß signaling pathway at the onset of BA delayed biliary degeneration.

American eels produce and release bile acid profiles that vary across life stage.

The American eel (Anguilla rostrata) is an imperilled fish hypothesized to use conspecific cues, in part, to coordinate long-distance migration during their multistage life history. Here, holding water and tissue from multiple American eel life stages was collected and analysed for the presence, profile and concentration of bile acids. Distinct bile acid profiles were identified in glass, elver, yellow eel and silver eel holding waters using ultraperformance liquid chromatography high-resolution mass spectrometry and principal component analysis. Taurochenodeoxycholic acid, taurodeoxycholic acid, cholic acid, deoxycholic acid, taurolithocholic acid and taurocholic acid were detected in whole tissue of American glass eels and elvers, and in liver, intestine and gallbladder samples of late-stage yellow eels. Bile acids were not a major component of silver eel washings or tissue. This study is novel because little was previously known about bile acids produced and emitted into the environment by American eels. Future behavioural studies could evaluate whether any bile acids produced by American eels influence conspecific migratory behaviour.

Sea lamprey cardiac mitochondrial bioenergetics after exposure to TFM and its metabolites.

Population control of invasive sea lamprey relies heavily on lampricide treatment of infested streams. The lampricide 3-trifluoromethyl-4-nitrophenol (TFM) is thought to impair mitochondrial ATP production through uncoupling oxidative phosphorylation. However, the effect of TFM on the entire electron transport chain (complexes I to V) in the mitochondria is not clear. In addition, TFM is reduced in phase I metabolism by sea lamprey at higher levels than in other fish species. The effects of these TFM reductive metabolites on mitochondria have not been explored. In this study, we sought to examine the effects of TFM and its reductive metabolite amino-TFM (TFMa) on cardiac mitochondrial oxygen consumption and membrane potential to delineate potential mechanisms for toxicity. To determine if molecules with similar structure also exhibit similar effects on mitochondria, we used 4-nitro-3-methylphenol (NMP) and its reductive metabolites 4-amino-3-methylphenol (NMPa) and 4-nitroso-3-methylphenol (NMPn) for comparisons. We found that mitochondrial bioenergetics was heavily affected with increasing concentrations of TFM, NMP, and NMPa when complexes I and II of the electron transport chain were examined, indicating that the toxic action of these compounds was exerted not only by uncoupling complex V, but also affecting complexes I and II.

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.

An Ancient CFTR Ortholog Informs Molecular Evolution in ABC Transporters.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel central to the development of secretory diarrhea and cystic fibrosis. The oldest CFTR ortholog identified is from dogfish shark, which retains similar structural and functional characteristics to the mammalian protein, thereby highlighting CFTR's critical role in regulating epithelial ion transport in vertebrates. However, the identification of an early CFTR ortholog with altered structure or function would provide critical insight into the evolution of epithelial anion transport. Here, we describe the earliest known CFTR, expressed in sea lamprey (Petromyzon marinus), with unique structural features, altered kinetics of activation and sensitivity to inhibition, and altered single-channel conductance compared to human CFTR. Our data provide the earliest evolutionary evidence of CFTR, offering insight regarding changes in gene and protein structure that underpin evolution from transporter to anion channel. Importantly, these data provide a unique platform to enhance our understanding of vertebrate phylogeny over a critical period of evolutionary expansion.

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.

Genomic sequence analyses of classical and non-classical lamprey progesterone receptor genes and the inference of homologous gene evolution in metazoans.

BACKGROUND: Nuclear progesterone receptor (nPR) is an evolutionary innovation in vertebrates that mediates genomic responses to progesterone. Vertebrates also respond to progesterone via membrane progesterone receptors (mPRs) or membrane associated progesterone receptors (MAPRs) through rapid nongenomic mechanisms. Lampreys are extant agnathan vertebrates, residing at the evolutionary juncture where vertebrates diverged from invertebrates. A survey of the progesterone receptor (PR) gene sequences in lamprey genomes would inform PR gene evolutionary events during the transition from invertebrates to vertebrates. RESULTS: In this study, we annotated sequences of one nPR, four mPR (ß, γ, δ and ε) and four MAPR genes from genomes of two lamprey species (Petromyzon marinus and Lethenteron japonicum). To infer the origin and evolutionary history of PR genes, we constructed phylogenetic trees of PR homologous sequences across representative species of metazoans. Phylogenetic analyses revealed that the mPRγ gene first appeared in non-bilaterians, and the mPRß gene likely arose from a duplication of mPRγ. On the other hand, the mPRγ gene gave rise to the mPRδ and ε genes much later in the vertebrate lineage. In addition, the mPRα gene first appeared in cartilaginous fishes, likely derived from duplication of mPRß after the agnathan-gnathostome divergence. All known MAPR genes were present in the lamprey genomes. Progesterone receptor membrane component 1 (PGRMC1), neudesin and neuferricin genes probably evolved in parallel in non-bilaterians, whereas two copies of PGRMC genes probably derived from duplication of ancestral PGRMC1 sequence and appeared before the speciation of lampreys. CONCLUSIONS: Non-classical mPR and MAPR genes first evolved in non-bilaterians and classical nPR genes evolved later in basal vertebrates. Sequence repertoires for membrane progesterone receptor genes in vertebrates likely originated from an ancestral metazoan sequence and expanded via several duplication events.

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.

High-sensitivity determination of estrogens in fish plasma using chemical derivatization upstream UHPLC-MSMS.

This article describes the development and validation of a sensitive LC-MSMS method for determination of estrogen in fish plasma. Dansyl chloride derivatization of the phenol functional group in estrogen was used to enhance the response to atmospheric pressure ionization leading to improve the sensitivity. Individual 13C internal standards were selected after comparison with deuterated standards. Liquid-liquid extraction (ethyl acetate or methyl tert-butyl ether) and protein precipitation (acetonitrile, methanol or acetone) were compared for the extraction and clean-up of estrogens from fish plasma. Ethyl acetate was selected as the best alternative with recovery ranging from 61 to 96% and matrix effect ranging from 88 to 106%. Limits of quantification ranged from 0.5 to 1pg/mL showing a gain in sensitivity of 10,000 times over electrospray ionization of underivatized estrogens. Accuracy and precision were validated over three consecutive days and the method was applied to measure estrogen in sea lamprey (Petromyzon marinus) and lake trout (Salvelinus namaycush) plasma. Estrone and estriol were detected in fish below 1ng/mL in plasma, justifying the need of a highly sensitive LC-MSMS quantification method.

A validated LC-MS/MS method for thyroid hormone determination in sea lamprey (Petromyzon marinus) plasma, gill, kidney and liver.

A UPLC-MS/MS method was developed to provide a reproducible, sensitive and quantitative assay to determine thyroid hormones in sea lamprey tissues and plasma. l-Thyroxine (T4) and its two triiodo-thyronine isomers have been simultaneously quantified and validated for plasma, gill, liver, and kidney matrices. Multiple sample preparation techniques were investigated to achieve optimal sample matrix digestion and clean-up. Enzymatic digestion followed by protein precipitation was selected to process the samples. The developed method exhibited excellent linearity for all analytes with regression coefficients higher than 0.99 for concentrations ranged from 10 to 50,000pg/mL. The limit of detection (LOD) was under 1pg/mL while the limit of quantification (LOQ) was estimated as 10pg/mL. This method was validated according to the FDA guidance and applied to determine thyroid hormone levels in plasma, gill and kidney of sea lamprey exposed to a sex pheromone. With appropriate implementation and further validation, this method could be applied to tissues in other species including humans.

Quantification of Oxidized and Unsaturated Bile Alcohols in Sea Lamprey Tissues by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry.

A sensitive and reliable method was developed and validated for the determination of unsaturated bile alcohols in sea lamprey tissues using liquid-liquid extraction and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The liver, kidney, and intestine samples were extracted with acetonitrile and defatted by n-hexane. Gradient UHPLC separation was performed using an Acquity BEH C18 column with a mobile phase of water and methanol containing 20 mM triethylamine. Multiple reaction monitoring modes of precursor-product ion transitions for each analyte was used. This method displayed good linearity, with correlation coefficients greater than 0.99, and was validated. Precision and accuracy (RSD %) were in the range of 0.31%-5.28%, while mean recoveries were between 84.3%-96.3%. With this technique, sea lamprey tissue samples were analyzed for unsaturated bile alcohol analytes. This method is practical and particularly suitable for widespread putative pheromone residue analysis.

Ultra-performance liquid chromatography tandem mass spectrometry for simultaneous determination of natural steroid hormones in sea lamprey (Petromyzon marinus) plasma and tissues.

This study aims to provide a rapid, sensitive and precise UPLC-MS/MS method for target steroid quantitation in biological matrices. We developed and validated an UPLC-MS/MS method to simultaneously determine 16 steroids in plasma and tissue samples. Ionization sources of Electrospray Ionization (ESI) and Atmospheric Pressure Chemical Ionization (APCI) were compared in this study by testing their spectrometry performances at the same chromatographic conditions, and the ESI source was found up to five times more sensitive than the APCI. Different sample preparation techniques were investigated for an optimal extraction of steroids from the biological matrices. The developed method exhibited excellent linearity for all analytes with regression coefficients higher than 0.99 in broad concentration ranges. The limit of detection (LOD) was from 0.003 to 0.1ng/mL. The method was validated according to FDA guidance and applied to determine steroids in sea lamprey plasma and tissues (fat and testes) by the developed method.