Preparation of Enzyme-Soluble Swim Bladder Collagen from Sea Eel (Muraenesox cinereus) and Evaluation Its Wound Healing Capacity.

In the present research, the enzyme-facilitated collagen from sea eel (Muraenesox cinereus) swim bladder was isolated, and the collagen characteristics were analyzed. Then, the collagen sponge was prepared and its potential mechanism in promoting skin wound healing in mice was further investigated. Collagen was obtained from the swim bladder of sea eels employing the pepsin extraction technique. Single-factor experiments served as the basis for the response surface method (RSM) to optimize pepsin concentration, solid-liquid ratio, and hydrolysis period. With a pepsin concentration of 2067 U/g, a solid-liquid ratio of 1:83 g/mL, and a hydrolysis period of 10 h, collagen extraction achieved a yield of 93.76%. The physicochemical analysis revealed that the extracted collagen belonged to type I collagen, and the collagen sponge displayed a fibrous structure under electron microscopy. Furthermore, in comparison to the control group, mice treated with collagen sponge dressing exhibited elevated activities of superoxide dismutase (SOD), catalase (CAT), total antioxidant capacity (T-AOC), and glutathione peroxidase (GSH-Px), and decreased levels of malondialdehyde (MDA), interleukin (IL)-1ß, interleukin (IL)-6, and tumor necrosis factor (TNF)-α. The collagen sponge dressing effectively alleviated inflammation in the wound area, facilitating efficient repair and rapid healing of the skin tissue. During the initial phase of wound healing, the group treated with collagen sponge dressing exhibited an enhancement in the expressions of cluster of differentiation (CD)31, epidermal growth factor (EGF), transforming growth factor (TGF)-ß1, and type I collagen, leading to an accelerated rate of wound healing. In addition, this collagen sponge dressing could also downregulate the expressions of CD31, EGF, and type I collagen to prevent scar formation in the later stage. Moreover, this collagen treatment minimized oxidative damage and inflammation during skin wound healing and facilitated blood vessel formation in the wound. Consequently, it exhibits significant potential as an ideal material for the development of a skin wound dressing.

Neuropeptide B (NPB) and NPB receptor 2b (NPBWR2b) in the ricefield eel Monopterus albus: expression and potential involvement in the regulation of gonadotropins.

The neuropeptide B/W signaling system is composed of neuropeptide B (NPB), neuropeptide W (NPW), and two cognate receptors, NPBWR1 and NPBWR2, which are involved in diverse physiological processes, including the central regulation of neuroendocrine axes in vertebrates. The components of this signaling system are not well conserved during vertebrate evolution, implicating its functional diversity. The present study characterized the ricefield eel neuropeptide B/W system, generated a specific antiserum against the neuropeptide B/W receptor, and examined the potential roles of the system in the regulation of adenohypophysial functions. The ricefield eel genome contains npba, npbb, and npbwr2b but lacks the npw, npbwr1, and npbwr2a genes. The loss of npw and npbwr1 probably occurred at the base of ray-finned fish radiation and that of npbwr2a species specifically in ray-finned fish. Npba and npbb genes are produced through whole-genome duplication (WGD) in ray-finned fish. The ricefield eel npba was expressed in the brain and some peripheral tissues, while npbb was predominantly expressed in the brain. The ricefield eel npbwr2b was also expressed in the brain and in some peripheral tissues, such as the pituitary, gonad, heart, and eye. Immunoreactive Npbwr2b was shown to be localized to Lh and Fsh cells but not to Gh or Prl cells in the pituitary of ricefield eels. Npba upregulated the expression of fshb and cga but not lhb mRNA in pituitary fragments of ricefield eels cultured in vitro. The results of the present study suggest that the NPB system of ricefield eels may be involved in the neuroendocrine regulation of reproduction.

Specific Signal Transduction of Constitutively Activating (D576G) and Inactivating (R476H) Mutants of Agonist-Stimulated Luteinizing Hormone Receptor in Eel.

We investigated the mechanism of signal transduction using inactivating (R476H) and activating (D576G) mutants of luteinizing hormone receptor (LHR) of eel at the conserved regions of intracellular loops II and III, respectively, naturally occurring in mammalian LHR. The expression of D576G and R476H mutants was approximately 58% and 59%, respectively, on the cell surface compared to those of eel LHR-wild type (wt). In eel LHR-wt, cAMP production increased upon agonist stimulation. Cells expressing eel LHR-D576G, a highly conserved aspartic acid residue, exhibited a 5.8-fold increase in basal cAMP response; however, the maximal cAMP response by high-agonist stimulation was approximately 0.62-fold. Mutation of a highly conserved arginine residue in the second intracellular loop of eel LHR (LHR-R476H) completely impaired the cAMP response. The rate of loss in cell-surface expression of eel LHR-wt and D576G mutant was similar to the agonist recombinant (rec)-eel LH after 30 min. However, the mutants presented rates of loss higher than eel LHR-wt did upon rec-eCG treatment. Therefore, the activating mutant constitutively induced cAMP signaling. The inactivating mutation resulted in the loss of LHR expression on the cell surface and no cAMP signaling. These data provide valuable information regarding the structure-function relationship of LHR-LH complexes.

Transcription factors Dmrt1a, Foxl2, and Nr5a1a potentially interact to regulate cyp19a1a transcription in ovarian follicles of ricefield eel (Monopterus albus).

Aromatase (encoded by Cyp19a1) in the ovarian follicular cells catalyzes the production of estradiol from testosterone, which plays important roles in the ovarian development of vertebrates. In the present study, the interaction of Dmrt1, Foxl2, and Nr5a1a on the regulation of cyp19a1a transcription in ovarian follicles was examined in a teleost, the ricefield eel Monopterus albus. The expression of dmrt1a, foxl2, and nr5a1a was detected in ovarian follicular cells together with cyp19a1a at the mRNA and/or protein levels. Sequence analysis identified one conserved Foxo binding site in the proximal promoter region of ricefield eel cyp19a1a. Transient transfection assay showed that Foxl2 may bind to the conserved Foxo site to activate cyp19a1a transcription and act synergistically with Nr5a1a. Mutation of either the conserved Nr5a1 site or Foxo site abolished or significantly decreased the synergistic effects of Nr5a1a and Foxl2 on cyp19a1a transcription. The sequence between Region III and I-box of Nr5a1a was critical to this synergistic effect. Dmrt1a modulated the Foxl2- and Nr5a1a-induced activation of cyp19a1a transcription and their synergistic effects in a biphasic manner, with inhibitory roles observed at lower doses (10-50 ng) but release of the inhibition or even potentiating effects observed at higher doses (100-200 ng). Collectively, data of the present study suggest that the interaction of Dmrt1a, Foxl2, and Nr5a1a in the ovarian follicular cells may facilitate the adequate expression of cyp19a1a and the production of estradiol, and contribute to the development and maturation of ovarian follicles in ricefield eels and other vertebrates as well.

The N-Linked Glycosylation Site N191 Is Necessary for PKA Signal Transduction in Eel Follicle-Stimulating Hormone Receptor.

The follicle-stimulating hormone receptor (FSHR) contains several N-linked glycosylation sites in its extracellular region. We conducted the present study to determine whether conserved glycosylated sites in eel FSHR are necessary for cyclic adenosine monophosphate (cAMP) signal transduction. We used site-directed mutagenesis to induce four mutations (N120Q, N191Q, N272Q, and N288Q) in the N-linked glycosylation sites of eel FSHR. In the eel FSHR wild-type (wt), the cAMP response was gradually increased in a dose-dependent manner (0.01-1500 ng/mL), displaying a high response (approximately 57.5 nM/104 cells) at the Rmax level. Three mutants (N120Q, N272Q, and N288Q) showed a considerably decreased signal transduction as a result of high-ligand treatment, whereas one mutant (N191Q) exhibited a completely impaired signal transduction. The expression level of the N191Q mutant was only 9.2% relative to that of the eel FSHR-wt, indicating a negligible expression level. The expression levels of the N120Q and N272Q mutants were approximately 35.9% and 24% of the FSHG-wt, respectively. The N288Q mutant had an expression level similar to that of the eel FSHR-wt, despite the mostly impaired cAMP responsiveness. The loss of the cell surface agonist-receptor complexes was very rapid in the cells expressing eel FSHR-wt and FSHR-N288Q mutants. Specifically, the N191Q mutant was completely impaired by the loss of cell surface receptors, despite treatment with a high concentration of the agonist. Therefore, we suggest that the N191 site is necessary for cAMP signal transduction. This finding implies that the cAMP response, mediated by G proteins, is directly related to the loss of cell surface receptors as a result of high-agonist treatment.

Cyt-C Mediated Mitochondrial Pathway Plays an Important Role in Oocyte Apoptosis in Ricefield Eel (Monopterus albus).

Apoptosis plays a key role in the effective removal of excessive and defective germ cells, which is essential for sequential hermaphroditism and sex change in vertebrates. The ricefield eel, Monopterus albus is a protogynous hermaphroditic fish that undergoes a sequential sex change from female to male. Previous studies have demonstrated that apoptosis is involved in sex change in M. albus. However, the apoptotic signaling pathway is unclear. In the current study, we explored the underlying mechanism of apoptosis during gonadal development and focused on the role of the mitochondrial apoptosis signaling pathway in sex change in M. albus. Flow cytometry was performed to detect apoptosis in gonads at five sexual stages and ovary tissues exposed to hydrogen peroxide (H2O2) in vitro. Then the expression patterns of key genes and proteins in the mitochondrial pathway, death receptor pathway and endoplasmic reticulum (ER) pathway were examined. The results showed that the apoptosis rate was significantly increased in the early intersexual stage and then decreased with the natural sex change from female to male. Quantitative real-time PCR revealed that bax, tnfr1, and calpain were mainly expressed in the five stages. ELISA demonstrated that the relative content of cytochrome-c (cyt-c) in the mitochondrial pathway was significantly higher than that of caspase8 and caspase12, with a peak in the early intersexual stage, while the levels of caspase8 and caspase12 peaked in the late intersexual stage. Interestingly, the Pearson's coefficient between cyt-c and the apoptosis rate was 0.705, which suggests that these factors are closely related during the gonadal development of M. albus. Furthermore, the cyt-c signal was found to be increased in the intersexual stage by immunohistochemistry. After incubation with H2O2, the mRNA expression of mitochondrial pathway molecules such as bax, apaf-1, and caspase3 increased in ovary tissues. In conclusion, the present results suggest that the mitochondrial apoptotic pathway may play a more important role than the other apoptotic pathways in sex change in M. albus.

The isosmotic point as critical salinity limit for growth and osmoregulation, but not survival, in the wolf eel Anarrhichthys ocellatus.

Members of wolf fish family Anarhichadidae have emerged as potential cold-water marine aquaculture species. This study examined growth performance and osmoregulation in juvenile wolf eel (Anarrhichthys ocellatus) held in a series of dilute salinities (30, 14, 9, and 6 ‰) over an 8-week trial. At the conclusion of the growth study, fish were sampled for analysis of gill and intestine enzyme activity, plasma ion content, and muscle moisture. Growth rate remained positive in all salinities throughout the 8-week trial. Specific growth rate was maintained above 3.0% mass day-1 at salinities of 30 and 14 ‰, but was significantly reduced at 9 (2.9% mass day-1) and 6 ‰ (2.0% mass day-1). Muscle water content increased with increasing salinity dilution (77.9% water in 30 ‰; 79.8% water in 6 ‰), and plasma osmolality (~ 320 mOsm kg-1) was maintained in salinities as dilute as 9 ‰ but was significantly lower (~ 280 mOsm kg-1) in the most dilute salinity of 6 ‰. Segmental linear regression analyses revealed that the calculated isosmotic point for wolf eel of ~ 10.6 ‰ was a critical limit for maintaining growth performance and osmoregulatory homeostasis. It is an important finding that fish considered to be a typical marine stenohaline organism could maintain ion and water balance as low as the isosmotic point, and exhibit survival and positive growth rates in salinities as dilute as 6 ‰. This work delivers a fundamental step in the empirical examination of this emerging aquaculture species and provides a model for evaluating osmoregulatory performance of marine stenohaline fishes in low-salinity aquaculture.

Metabonomic Insights into the Sperm Activation Mechanisms in Ricefield Eel (Monopterus albus).

In fish, sperm motility activation is one of the most essential procedures for fertilization. Previous studies have mainly focused on the external environmental effects and intracellular signals in sperm activation; however, little is known about the metabolic process of sperm motility activation in fish. In the present study, using ricefield eel (Monopterus albus) sperm as a model, metabonomics was used to analyze the metabolic mechanism of the sperm motility activation in fish. Firstly, 529 metabolites were identified in the sperm of ricefield eel, which were clustered into the organic acids, amino acids, nucleotides, benzene, and carbohydrates, respectively. Among them, the most abundant metabolites in sperm were L-phenylalanine, DL-leucine, L-leucine, lysolecithin choline 18:0, L-tryptophan, adenine, hypoxanthine, 7-Methylguanine, shikimic acid, and L-tyrosine. Secondly, compared to pre-activated sperm, the level of S-sulfo-L-cysteine and L-asparagine were both increased in the post-activated sperm. Ninety-two metabolites were decreased in the post-activated sperm, including quinic acid, acetylsalicylic acid, 7,8-dihydro L-biopterin, citric acid, glycylphenylalanine, and dihydrotachysterol (DHT). Finally, basing on the pathway analysis, we found that the changed metabolites in sperm motility activation were mainly clustered into energy metabolism and anti-oxidative stress. Fish sperm motility activation would be accompanied by the release of a large amount of energy, which might damage the genetic material of sperm. Thus, the anti-oxidative stress function is a critical process to maintain the normal physiological function of sperm.

Swimming under elevated hydrostatic pressure increases glycolytic activity in gas gland cells of the European eel.

In spite of many decades of research, the spawning migration of the European eel Anguilla anguilla from the European coast to the Sargasso Sea remains a mystery. In particular, the role of the swimbladder as a buoyancy regulating structure is not yet understood. In this study, we exercised silver eels in a swim tunnel under elevated hydrostatic pressure. The transcriptome of gas gland tissue of these exercised eels was then compared to the known transcriptome of not exercised (control) silver eel gas gland cells. Due to the high infection rate of the eel population with the swimbladder parasite Anguillicola crassus, the comparison also included an exercised group of silver eels with a heavily damaged swimbladder, and we compared the previously published transcriptome of not exercised silver eels with a highly damaged swimbladder with the exercised group of silver eels with a heavily damaged swimbladder. The comparisons of unexercised (control) silver eels with exercised silver eels with functional swimbladder (EF), as well as with exercised silver eels with damaged swimbladder (ED), both showed a significant elevation in transcripts related to glycolytic enzymes. This could also be observed within the comparison of unexercised silver eels with a highly infected swimbladder with exercised eels with a damaged swimbladder (DED). In contrast to EF, in ED a significant elevation in transcript numbers of mitochondrial NADH dehydrogenase was observed. While in EF the transcriptional changes suggested that acid production and secretion was enhanced, in ED these changes appeared to be related to thickened tissue and thus elevated diffusion distances. The remarkable number of differentially expressed transcripts coding for proteins connected to cAMP-dependent signaling pathways indicated that metabolic control in gas gland cells includes cAMP-dependent pathways. In contrast to ED, in EF significant transcriptional changes could be related to the reconstruction of the extracellular matrix, while in ED tissue repair and inflammation was more pronounced. Surprisingly, in exercised eels hypoxia inducible transcription factor expression was elevated. In EF, a large number of genes related to the circadian clock were transcriptionally modified, which may be connected to the circadian vertical migrations observed during the spawning migration.

Differential distribution and potential regulatory roles of estrogen receptor 2a and 2b in the pituitary of ricefield eel Monopterus albus.

Estrogens play important regulatory roles in the pituitary of vertebrates. Two forms of estrogen receptor 2 (Esr2), namely Esr2a and Esr2b, are identified in teleosts, but their differential roles remain to be fully elucidated. In the present study, expression and potential functional roles of Esr2a and Esr2b were characterized in ricefield eels. esr2a and esr2b mRNA were broadly distributed in tissues, with high levels observed in the brain, pituitary, and gonads. In order to examine the cellular localization of Esr2a and Esr2b in the pituitary, specific antisera against ricefield eel Esr2a and Esr2b were generated, respectively. Interestingly, immunohistochemistry and Western blot analysis revealed that Esr2a and Esr2b were differentially distributed in the pituitary, with the former localized to the adenohypophysis while the latter to the neurohypophysis. Dual fluorescent immunostaining showed that immunoreactive Esr2a was present in Gh and Prl cells, but not in Lh and Fsh cells. Estradiol (E2) stimulated lhb and prl gene expression in dispersed pituitary cells of intersexual ricefield eels, but had no effects on gh, fshb, and gnrhr2 gene expression and Gh release. Results of the present study are helpful for further understanding the roles and mechanisms of estrogen signals in the pituitary.

Presence of CTXs in moray eels and dusky groupers in the marine environment of the Canary Islands.

Local population frequently consumes moray eels and dusky groupers from the Canary Islands. These species are top predators and the interactions between them include predation but also, in some cases, collaborative hunting. These fish are well known to cause ciguatera (CFP) outbreaks in several marine areas such as Japan, Hawaii, French Polynesia and Caribe. Groupers have been involved in CFP events in the Canary Islands, however, moray eels have not yet been well studied in this regard. The present research seeks to describe the finding of a black moray in the stomach of a positive dusky grouper during its necropsy, and to clarify the implication of groupers and moray eels in the food webs, accumulating CTXs in the Canarian environment. The study also updates statistics on the presence of toxic groupers in this archipelago. For these purposes, 248 grouper samples from the CFP official control in the Canary Islands (2018-2019) were analysed and 36 moray eels (5 species) were collected under the EuroCigua project and one was obtained during a dusky grouper necropsy. All samples were analysed with the Neuro-2a cell-based assay (CBA) to evidence CTX-like toxicity. Regarding the necropsied grouper and the moray eel found in its stomach content, the LCMS/MS method allowed the identification and quantification of CCTX1 in both fish at similar levels while none of the P-CTXs for which standards were available were detected. Among groupers, 25.4 % displayed CTX-like toxicity with differences between islands. For moray eels 38.9 % showed toxicity, involving 4 species. Black moray exhibited a high proportion of positives (9/12) and a positive correlation was found between CTX-like toxicity quantification and the black moray weight. Regarding the grouper, and the moray eel found in its stomach, the LCMS/MS method allowed the identification and quantification of C-CTX1 in both fish at similar levels. This found suggests a trophic interaction between these species and their role in maintaining CTXs in the Canary waters where local population commonly demand those species for consumption. The island of El Hierro stands out above all the other Canary Islands with the concerning percentage of positive grouper samples and the high CTX toxicity levels obtained in moray eel specimens analysed in this marine area. This is the first report of CTX-like toxicity in flesh of moray eels fished in the Canary archipelago and the confirmation of the presence of C-CTX1 by LCMS/MS in a black moray from this marine area.

Estrogen suppresses bcl2 expression through dnmt1 in Japanese eel ovarian follicle.

Japanese eels are commercially valuable species in Asian aquaculture. This study evaluated whether salmon pituitary extract (SPE) or 17ß-estradiol (E2) treatment can induce cytotoxic activity in eel ovarian follicles. Follicular cells died after exposure SPE for 24-h culture in an in vitro culture. Moreover, the E2 treatment also significantly reduced follicular cell counts. These results reveal that the inhibition of follicular cell numbers by SPE may occur through SPE-induced steroidogenesis. Results of a quantitative PCR analysis indicated that adding E2 to the culture decreased bcl2 and increased dnmt1 mRNA expression in Japanese eel follicular cells after 24 h. The results of a promoter assay revealed that E2 significantly increase dnmt1 promoter activity through estrogen receptor-binding site. An in silico analysis predicted several putative transcription factors targeting the bcl2 gene promoter region. Methylation of the bcl2 promoter accounted for the downregulation of bcl2 by E2-mediated dnmt1. The DNA methylation level of the bcl2 gene was significantly higher in E2-treated follicular cells than that in the control group. Finally, the E2-induced hypermethylation pattern of the bcl2 promoter and the reduction in follicular cell numbers were suppressed by adding an MTase inhibitor. Our findings demonstrate that estrogen has a negative effect on the reproductive system of female eels by regulating an epigenetic mechanism during artificial maturation.

Selective Depletion of Chiral 4-Hydroxypraziquantel Metabolites in Three Types of Aquaculture Fish by LC-MS/MS.

The rise of aquaculture has necessitated the use of antibiotics and other agents in these densely populated and often closed environments. An enantioselective depletion study of four chiral 4-hydroxypraziquantel metabolites (4-OH-PZQ) in perch, tilapia, and ricefield-eel muscles was done using a simple, sensitive, and reliable liquid-chromatography-tandem-mass-spectrometry (LC-MS/MS) method. These metabolites result from the uptake of the drug praziquantel (PZQ), which is used in aquaculture. A novel strategy of using a C18 short column in tandem with a chiral hydroxypropyl-ß-cyclodextrin superficially porous particle (CDShell-RSP) column produced the optimal separation for both the enantiomers and diastereoisomers of 4-OH-PZQ. The method was linear over the concentration range of 1-250 µg L-1 ( r2 ≥ 0.99) for R- trans-4-OH-PZQ, S- trans-4-OH-PZQ, R- cis-4-OH-PZQ, and S- cis-4-OH-PZQ. The average recoveries of four analytes at three spiked levels of 1, 10, and 100 µg kg-1 ranged from 84.2 to 93.1%, and the intraday and interday relative standard deviations were less than 7.9%. The limits of quantification of the four 4-OH-PZQ metabolites in perch-, tilapia-, and ricefield-eel-muscle matrices were 1.0 µg kg-1. The method was utilized to monitor the depletion of trans- and cis-4-OH-PZQ enantiomers in perch, tilapia, and ricefield-eel muscle following oral administration (medicine bath for ricefield eel). Species-specific differences in the PZQ metabolism of isomers were observed. In addition, new metabolites of PZQ were observed: 3-hydroxypraziquantel diastereomers.

Site-specific roles of N-linked oligosaccharides in recombinant eel follicle-stimulating hormone for secretion and signal transduction.

Eel follicle-stimulating hormone (eelFSH) is composed of a common α-subunit and a hormone specific ß-subunit, both of which contain two N-linked carbohydrate residues. We characterized the biologically active single chains by fusing the α-subunit to the carboxyl terminal region of the eelFSH ß-subunit. Expression vectors were constructed and the biological activity of the recombinant hormones (rec-hormones) was characterized using Chinese hamster ovary (CHO) K1 cells expressing the eelFSH receptor gene. Mutagenesis of the individual and double glycosylated sites was performed to determine the functions of the oligosaccharide chains on signal transduction. The absence of the Asn22 (eelFSHßΔ22/α) and Asn5.22 (eelFSHßΔ5.22/α) N-linked oligosaccharide chain in the eelFSH ß-subunit completely reduced the secretion level in the medium and cell lysate of CHO-K1 cells. The expression levels of eelFSHß/α wild-type in CHO suspension (CHO-S) cells was approximately 4-fold higher in CHO-k1 cells. The molecular weight of rec-eelFSHß/α wild-type by western blotting analysis was found to be 34 kDa. Mutants (ß/αΔ56, ß/αΔ79, and ßΔ5/α) lacking single oligosaccharide sites showed molecular weights that were reduced by approximately 10%. The digestion of N-linked oligosaccharides using PNGaseF treatment showed that the molecular weights of all mutants were reduced to 27-kDa. The oligosaccharide chains in rec-eelFSHß/α wild-type were modified to a molecular weight of approximately 7-10 kDa in CHO-K1 and CHO-S cells. Oligosaccharide site deletions at positions Asn56 and Asn79 on the α-subunit and Asn5 on the ß-subunit were found to play an essential role in cAMP signal transduction through the eelFSH receptor. The EC50 values of Asn56 and Asn5 resulted in a significant decrease in potency to 64% and 53% of the wild type, respectively. Specifically, the removal of the carbohydrates at Asn79 of the α-subunit (ß/αΔ79) was drastically reduced to 53.8% of the wild-type levels in maximum response. These results have allowed for the identification of the site-specific roles of carbohydrate residues in eel FSH. Our data suggest that N-linked oligosaccharide chains play a pivotal role in biological activity through the eelFSH receptor as suggested in similar studies of other mammalian FSH hormones.

The expression of nuclear and membrane estrogen receptors in the European eel throughout spermatogenesis.

Estradiol (E2) can bind to nuclear estrogen receptors (ESR) or membrane estrogen receptors (GPER). While mammals possess two nuclear ESRs and one membrane GPER, the European eel, like most other teleosts, has three nuclear ESRs and two membrane GPERs, as the result of a teleost specific genome duplication. In the current study, the expression of the three nuclear ESRs (ESR1, ESR2a and ESR2b) and the two membrane GPERs (GPERa and GPERb) in the brain-pituitary-gonad (BPG) axis of the European eel was measured, throughout spermatogenesis. The eels were first transferred from freshwater (FW) to seawater (SW), inducing parallel increases in E2 plasma levels and the expression of ESRs. This indicates that salinity has a stimulatory effect on the E2 signalling pathway along the BPG axis. Stimulation of sexual maturation by weekly injections of human chorionic gonadotropin (hCG) induced a progressive decrease in E2 plasma levels, and different patterns of expression of ESRs and GPERs in the BPG axis. The expression of nuclear ESRs increased in some parts of the brain, suggesting a possible upregulation due to a local production of E2. In the testis, the highest expression levels of the nuclear ESRs were observed at the beginning of spermatogenesis, possibly mediating the role of E2 as spermatogonia renewal factor, followed by a sharply decrease in the expression of ESRs. Conversely, there was a marked increase observed in the expression of both membrane GPERs throughout spermatogenesis, suggesting they play a major role in the final stages of spermatogenesis.

The European Eel NCCß Gene Encodes a Thiazide-resistant Na-Cl Cotransporter.

The thiazide-sensitive Na-Cl cotransporter (NCC) is the major pathway for salt reabsorption in the mammalian distal convoluted tubule. NCC plays a key role in the regulation of blood pressure. Its inhibition with thiazides constitutes the primary baseline therapy for arterial hypertension. However, the thiazide-binding site in NCC is unknown. Mammals have only one gene encoding for NCC. The eel, however, contains a duplicate gene. NCCα is an ortholog of mammalian NCC and is expressed in the kidney. NCCß is present in the apical membrane of the rectum. Here we cloned and functionally characterized NCCß from the European eel. The cRNA encodes a 1043-amino acid membrane protein that, when expressed in Xenopus oocytes, functions as an Na-Cl cotransporter with two major characteristics, making it different from other known NCCs. First, eel NCCß is resistant to thiazides. Single-point mutagenesis supports that the absence of thiazide inhibition is, at least in part, due to the substitution of a conserved serine for a cysteine at position 379. Second, NCCß is not activated by low-chloride hypotonic stress, although the unique Ste20-related proline alanine-rich kinase (SPAK) binding site in the amino-terminal domain is conserved. Thus, NCCß exhibits significant functional differences from NCCs that could be helpful in defining several aspects of the structure-function relationship of this important cotransporter.

Functional Characterisation of Eel Dopamine D2 Receptors and Involvement in the Direct Inhibition of Pituitary Gonadotrophins.

In various vertebrate species, dopamine (DA) exerts an inhibitory action on reproduction. In the European eel, DA plays a pivotal role in the inhibitory control of gonadotroph function and the blockade of puberty. In vivo studies have suggested that this effect is mediated by receptors pharmacologically related to the D2 family. In the European eel, two distinct D2 receptor (D2-R) paralogous genes have been identified (D2A-R and D2B-R) and both were shown to be expressed in the pituitary. We investigated the potential role of each paralogue in the control of gonadotroph function in this species. Eel recombinant D2A-R or D2B-R were expressed in HEK 293 cells, with a universal Gα subunit, and receptor activation was followed by inositol phosphate production. Recombinant D2-Rs exhibited a comparable affinity for DA, although they had differential affinities for mammalian D2-R agonists and antagonists, supporting subtle structure/activity differences. Furthermore, using eel pituitary cell primary cultures, the expression by gonadotroph cells of both native eel D2-R paralogues was examined by in situ hybridisation of D2A-R or D2B-R transcripts, coupled with immunofluorescence of luteinising hormone (LH)ß or follicle-stimulating (FSH)ß. LH and to a lesser extent, FSH cells expressed both D2-R transcripts but with a clear predominance of D2B-R. Notably, D2B-R transcripts were detected for the majority of LH cells. Accordingly, using these cultures, we showed that DA potently inhibited basal and testosterone-stimulated LHß expression and less potently basal and activin-stimulated FSHß expression. We also tested some D2-R antagonists, aiming to select the most adequate one to be used in innovative protocols for induction of eel sexual maturation. We identified eticlopride as the most potent inhibitor of DA action on basal and stimulated LH expression in vitro. Our data suggest a differential functionalisation of the duplicated receptor genes and demonstrate that mainly D2B-R is involved in the dopaminergic inhibitory control of eel gonadotroph function.

Duplicated CFTR isoforms in eels diverged in regulatory structures and osmoregulatory functions.

Two cystic fibrosis transmembrane conductance regulator (CFTR) isoforms, CFTRa and CFTRb, were cloned in Japanese eel and their structures and functions were studied in different osmoregulatory tissues in freshwater (FW) and seawater (SW) eels. Molecular phylogenetic results suggested that the CFTR duplication in eels occurred independently of the duplication event in salmonid. CFTRa was expressed in the intestine and kidney and downregulated in both tissues in SW eels, while CFTRb was specifically expressed in the gill and greatly upregulated in SW eels. Structurally, the CFTR isoforms are similar in most functional domains except the regulatory R domain, where the R domain of CFTRa is similar to that of human CFTR but the R domain of CFTRb is unique in having high intrinsic negative charges and fewer phosphorylation sites, suggesting divergence of isoforms in terms of gating properties and hormonal regulation. Immunohistochemical results showed that CFTR was localized on the apical regions of SW ionocytes, suggesting a Cl(-) secretory role as in other teleosts. In intestine and kidney, however, immunoreactive CFTR was mostly found in the cytosolic vesicles in FW eels, indicating that Cl(-) channel activity could be low at basal conditions, but could be rapidly increased by membrane insertion of the stored channels. Guanylin (GN), a known hormone that increases CFTR activity in mammalian intestine, failed to redistribute CFTR and to affect its expression in eel intestine. The results suggested that GN-independent CFTR regulation is present in eel intestine and kidney.

Scp3 expression in relation to the ovarian differentiation in the protogynous hermaphroditic ricefield eel Monopterus albus.

Synaptonemal complex protein 3 (Scp3), which is encoded by scp3, is a meiotic marker commonly used to trace the timing of gonadal differentiation in vertebrates. In the present study, the ricefield eel scp3 cDNA was cloned, and a fragment encoding amino acids 49 to 244 was overexpressed. The recombinant Scp3 polypeptide was purified and used to generate a rabbit anti-Scp3 polyclonal antiserum. In adult ricefield eels, scp3 mRNA was predominantly detected in the gonads and faintly detected in discrete brain areas. In the gonads, Scp3 immunoreactivities were shown to be localized to the germ cells, including meiotic primary growth oocytes, spermatocytes, and pre-meiotic spermatogonia. During early ovarian differentiation, immunoreactive Scp3 was not detected in the gonads of ricefield eels at 6 days post-hatching (dph) but was found to be abundantly localized in the cytoplasm of some oogonia at 7 dph, coinciding with the appearance of the ovarian cavity and ovarian differentiation. At 14 dph, strong Scp3 immunostaining was detected on one side of the nucleus with a distinct polarity in some germ cells, presumably at the leptotene stage. Consistent with these results, the expression of scp3 mRNA was faintly detected in the gonads of ricefield eels at 6 dph, increased at 8 dph, and then remained relatively high thereafter. Taken together, these results suggest that the appearance of immunoreactive Scp3 in oogonia could be a marker for early ovarian differentiation in ricefield eels. The translocation of the Scp3 protein from the cytoplasm to the nucleus in the oogonium of ricefield eels appears to be a controlled process that warrants further study.

Pharmacokinetics and residue depletion of praziquantel in rice field eels Monopterus albus.

We investigated the pharmacokinetic characteristics of praziquantel (PZQ) in rice field eels Monopterus albus. Pharmacokinetic parameters were determined following a single intravenous administration (5 mg kg(-1) body weight [bw]) and a single oral administration (10 mg kg(-1) bw) at 22.0 ± 0.7°C. We also evaluated residue depletion in tissues following daily administration of PZQ (10 mg kg(-1) bw) that was given orally for 3 consecutive days at 22.0 ± 0.7°C. Following intravenous treatment, the plasma concentration-time curve was best described by a 3-compartment open model, with distribution half-life (t(1/2α)), elimination half-life (t(1/2ß)), and area under the concentration-time curve (AUC) of 0.54 h, 17.10 h, and 14505.12 h µg l(-1), respectively. After oral administration, the plasma concentration-time curve was best described by a 1-compartment open model with first-order absorption, with absorption half-life (t(1/2Ka)), elimination half-life (t(1/2Ke)), peak concentration (C(max)), time-to-peak concentration (T(max)), and AUC estimated to be 2.28 h, 6.66 h, 361.29 µg l(-1), 5.36 h, and 6065.46 h µg l(-1), respectively. The oral bioavailability (F) was 20.9%. With respect to residue depletion of PZQ, the t(1/2ß) values of muscle, skin, liver, and kidney were 20.2, 28.4, 14.9, and 54.1 h, respectively. Our results indicated rapid absorption, rapid elimination, and low bioavailability of PZQ in rice field eels at the tested dosing conditions.