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.

Characterization of Somatically-Eliminated Genes During Development of the Sea Lamprey (Petromyzon marinus).

The sea lamprey (Petromyzon marinus) is a basal vertebrate that undergoes developmentally programmed genome rearrangements (PGRs) during early development. These events facilitate the elimination of ∼20% of the genome from the somatic cell lineage, resulting in distinct somatic and germline genomes. Thus far only a handful of germline-specific genes have been definitively identified within the estimated 500 Mb of DNA that is deleted during PGR, although a few thousand germline-specific genes are thought to exist. To improve our understanding of the evolutionary/developmental logic of PGR, we generated computational predictions to identify candidate germline-specific genes within a new transcriptomic dataset derived from adult germline and the early embryonic stages during which PGR occurs. Follow-up validation studies identified 44 germline-specific genes and further characterized patterns of transcription and DNA loss during early embryogenesis. Expression analyses reveal that many of these genes are differentially expressed during early embryogenesis and presumably function in the early development of the germline. Ontology analyses indicate that many of these germline-specific genes play known roles in germline development, pluripotency, and oncogenesis (when misexpressed). These studies provide support for the theory that PGR serves to segregate molecular functions related to germline development/pluripotency in order to prevent their potential misexpression in somatic cells. This larger set of eliminated genes also allows us to extend the evolutionary/developmental breadth of this theory, as some deleted genes (or their gnathostome homologs) appear to be associated with the early development of somatic lineages, perhaps through the evolution of novel functions within gnathostome lineages.

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.

An UPLC-MS/MS method for quantitative profiling of bile acids in sea lamprey plasma and tissues.

Bile acids (BAs) have recently gained more attention because of their diverse roles from digestion to signaling. Simultaneous analyses of various BAs in biological samples are challenging due to their structural similarity, relatively low concentrations, and the presence of isomeric forms. In this study, we report a simple and sensitive UPLC-MS/MS method for simultaneous quantifications of 13 BAs including four unique sea lamprey BAs in sea lamprey plasma, liver, intestine, and gills. A straightforward protein precipitation (PPT) method was used to extract BAs from the biological samples. Separation of all target analytes was achieved on a reverse-phase UPLC column in 15min, and detection was carried out on MS/MS with ESI in the negative ionization mode. This method was validated regarding its linearity, limits of detection (LOD), recovery, matrix effect, reproducibility, accuracy and precision. Significant improvements compared to previous LC-MS/MS methods were observed as a result of the application of UPLC and extensive optimization of experimental conditions. The method showed excellent linearity with high regression coefficients (>0.99) over a range of 0.5-1000ng/mL and LODs ranged from 0.009 to 0.11ng/mL. The applications of the developed method demonstrated that it simultaneously determined all target BAs in different biological sample matrices with excellent sensitivity, selectivity and reproducibility.

An anti-steroidogenic inhibitory primer pheromone in male sea lamprey (Petromyzon marinus).

Reproductive functions can be modulated by both stimulatory and inhibitory primer pheromones released by conspecifics. Many stimulatory primer pheromones have been documented, but relatively few inhibitory primer pheromones have been reported in vertebrates. The sea lamprey male sex pheromone system presents an advantageous model to explore the stimulatory and inhibitory primer pheromone functions in vertebrates since several pheromone components have been identified. We hypothesized that a candidate sex pheromone component, 7α, 12α-dihydroxy-5α-cholan-3-one-24-oic acid (3 keto-allocholic acid or 3kACA), exerts priming effects through the hypothalamic-pituitary-gonadal (HPG) axis. To test this hypothesis, we measured the peptide concentrations and gene expressions of lamprey gonadotropin releasing hormones (lGnRH) and the HPG output in immature male sea lamprey exposed to waterborne 3kACA. Exposure to waterborne 3kACA altered neuronal activation markers such as jun and jun N-terminal kinase (JNK), and lGnRH mRNA levels in the brain. Waterborne 3kACA also increased lGnRH-III, but not lGnRH-I or -II, in the forebrain. In the plasma, 3kACA exposure decreased all three lGnRH peptide concentrations after 1h exposure. After 2h exposure, 3kACA increased lGnRH-I and -III, but decreased lGnRH-II peptide concentrations in the plasma. Plasma lGnRH peptide concentrations showed differential phasic patterns. Group housing condition appeared to increase the averaged plasma lGnRH levels in male sea lamprey compared to isolated males. Interestingly, 15α-hydroxyprogesterone (15α-P) concentrations decreased after prolonged 3kACA exposure (at least 24h). To our knowledge, this is the only known synthetic vertebrate pheromone component that inhibits steroidogenesis in males.

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

BACKGROUND: Vertebrate 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. RESULTS: The results showed that natural male pheromone mixtures induced differential steroid responses but facilitated sexual maturation in both sexes of immature animals (χ(2) = 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. CONCLUSIONS: We conclude that a single male pheromone component primes the HPG axis in immature sea lamprey in a sexually dimorphic manner.

Annual sex steroid and other physiological profiles of Pacific lampreys (Entosphenus tridentatus).

We documented changes in plasma levels of estradiol 17-beta (E2), progesterone (P), 15alpha-hydroxytestosterone (15alpha-T), thyroxine (T4), triiodothyronine (T3), protein, triglycerides (TGs), and glucose in adult Pacific lampreys (Entosphenus tridentatus) held in the laboratory in two different years. Levels of E2 in both sexes ranged from 0.5 to 2 ng/mL from September to March, peaked in late April (2-4 ng/mL), and decreased in May, with levels higher in males than in females. Levels of P were low from September through April, but then increased substantially during May (2-4 ng/mL), with levels again highest in males. Levels of 15alpha-T in males were around 0.75 ng/mL through the winter before exceeding 1 ng/mL in April and decreasing thereafter, whereas females showed a gradual increase from 0.25 ng/mL in November to 0.5 ng/mL in April before decreasing. Thyroxine concentrations differed between fish in each year, with most having levels ranging from 0.75 to 2.5 ng/mL in the fall and winter, and only fish in 2003 showing distinct peaks (3-4 ng/mL) in early April or May. Plasma T3 was undetectable from November through mid-March before surging dramatically in April (ca. 150 ng/mL) and decreasing thereafter. Levels of protein, TGs, and glucose decreased or were stable during the fall and winter with TGs and glucose surging in late April to early May for some fish. Our study is the first to document long-term physiological changes in Pacific lampreys during overwintering and sexual maturation and increases our understanding of the life history of this unique fish.

Isolation of lysozyme and an antifungal peptide from sea lamprey (Petromyzon marinus) plasma.

The sea lamprey (Petromyzon marinus) belongs to the most primitive class of fish and has only innate immunity. The innate immune factors, lysozyme and an antifungal peptide, were isolated from sea lamprey plasma. Sea lamprey plasma (40.1mg protein/ml) was assayed for lysozyme activity by gel diffusion assay. Using hen egg white lysozyme standards, plasma concentration of lamprey lysozyme was 5microg lysozyme/mg total protein. The presence of lysozyme in such high concentration in lamprey plasma could be important in their innate immunity and resistance to infection. Lysozyme and the antifungal peptide were isolated by low molecular weight gel filtration chromatography from sea lamprey plasma. Gel filtration chromatography yielded two peak tubes containing lysozyme (1microg/211microg total protein) and antifungal peptide (1microg/66microg total protein). Lysozyme and antifungal activity of each fraction were determined by well diffusion assay using Gram-negative bacteria, Gram-positive bacteria and two fungal species. The molecular weight of lamprey lysozyme was 14.3kDa. The sea lamprey lysozyme was effective against Gram-positive bacteria but not against Gram-negative bacteria or fungi. Molecular weight of the antifungal peptide was approximately 3000Da. Antifungal plasma activity was seen against Penicillium notatum and Aspergillus flavus. No plasma antibacterial peptide was found.

Tribute to R. G. Boutilier: evidence of a high activity carbonic anhydrase isozyme in the red blood cells of an ancient vertebrate, the sea lamprey Petromyzon marinus.

Carbonic anhydrase (CA) is a multi-functional enzyme that catalyzes the hydration/dehydration of carbon dioxide. In the red blood cell (rbc), CA is necessary to facilitate the transport of carbon dioxide out of the body. Results from earlier biochemical studies indicate that ancient vertebrates, such as agnathans, possess a low activity rbc CA isozyme, whereas more recently evolved vertebrates, such as teleost fish, possess a high activity isozyme. At present, however, the changes in the molecular structure that have resulted in this large increase in catalytic efficiency are unknown. The objective of the current study was therefore to determine the molecular structure of rbc CA in lampreys and compare it to that of teleosts in an effort to ascertain how this important enzyme became more efficient over evolutionary time. Isolation and sequencing of cytoplasmic CA from rbc and gill showed only a single isozyme of 789 bp (262 amino acids). This isozyme was also found in brain and kidney, with no evidence of additional cytoplasmic CA isozymes in other tissues. Phylogenetic analysis grouped this isozyme closely to vertebrate CA VII, which is ancestral to the rbc isozymes in other vertebrates. Interestingly, active site analysis revealed a structure similar to high activity isozymes. A comparative kinetic analysis of CA from rbc lysates and CA fusion proteins showed that the traditional method of determining the turnover number may not be appropriate for all vertebrate CAs. In contrast to previous evidence, lamprey CA was found to be a high activity isozyme. These results suggest that the critical functional characteristics of rbc CA have been highly conserved throughout vertebrate evolution.