Apolipoprotein Complexity in Japanese Eel Anguilla japonica: Truncated Apolipoprotein A-I and Apolipoprotein A-I-like Protein in Plasma Lipoproteins

A truncated apolipoprotein (apo) A-I with a molecular weight (M _r) of 26 kDa was first isolated from the plasma high density lipoproteins of an atypical Japanese eel (Anguilla japonica). Interestingly, this eel contained a very small amount of intact apoA-I (M _r28 kDa) in the plasma, although serine protease inhibitors were present throughout the plasma preparation. The N-terminal sequence of 20 amino acids in truncated apoA-I was completely identical with that of intact apoA-I. Another apolipoprotein with M _r28 kDa, whose N-terminal amino acid sequence differed from apoA-I, was also found in high density lipoprotein and low density lipoprotein. The apolipoprotein profiles of Japanese eel plasma appear to be complicated.     

Rectal water absorption in seawater-adapted Japanese eel Anguilla japonica

Marine teleosts drink large amounts of seawater to compensate for continuous osmotic water loss. We investigated a possible significant role of the rectum in water absorption in seawater-adapted eel. In rectal sacs filled with balanced salt solution (BSS) and incubated in isotonic BSS, water absorption was greater in seawater-adapted eel than in freshwater eel. Since rectal fluid osmolality was slightly lower than plasma osmolality in seawater-adapted eel, effects of rectal fluid osmolality on water absorption were examined in rectal sacs filled with artificial rectal fluid with different osmolality. Rectal water absorption was greater at lower rectal fluid osmolality, suggesting that an osmotic gradient between the blood and rectal fluid drives the water movement. Ouabain, a specific inhibitor of Na⁺/K⁺-ATPase, inhibited water absorption in rectal sacs, indicating that an osmotic gradient favorable to rectal water absorption was created by ion uptake driven by Na⁺/K⁺-ATPase. Expression levels of aquaporin 1 (AQP1), a water-selective channel, were significantly higher in the rectum than in the anterior and posterior intestines. Immunoreaction for Na⁺/K⁺-ATPase was detected in the mucosal epithelial cells in the rectum with more intense staining in the basal half than in the apical half, whereas AQP1 was located in the apical membrane of Na⁺/K⁺-ATPase-immunoreactive epithelial cells. The rectum is spatially separated from the posterior intestine by a valve structure and from the anus by a sphincter. Such structures allow the rectum to swell as intestinal fluid flows into it, and a concomitant increase in hydrostatic pressure may provide an additional force for rectal water absorption. Our findings indicate that the rectum contributes greatly to high efficiency of intestinal water absorption by simultaneous absorption of ions and water.     

Population structure of the Japanese eel, Anguilla japonica

Mitochondrial DNA (mtDNA) sequences that include (a) a part of the cytochrome b gene, (b) two tRNA genes, and (c) a part of the noncoding D-loop region of 31 Anguilla japonica (Japanese eel) and 1 A. marmorata collected from Taiwan, Japan, and mainland China were determined to evaluate the population structure of Japanese eel. Among 30 genotypes identified from the 31 Japanese eel mtDNAs sequenced, there are 58 variable sites, predominantly clustered at the D-loop region. The phylogenetic tree constructed by the unweighted pair-group method with arithmetic mean shows neither significant genealogical branches nor geographic clusters. Furthermore, the sequence-statistics test reveals little, if any, significant genetic differentiation. These results indicate that the 31 Japanese eels might come from a single population. Analysis of sequence variation in mtDNA by using the relationship between the number of segregating sites and the average number of nucleotide differences under the neutral mutation hypothesis reveals that neutral mutation acts as a major factor influencing the evolutionary divergence of the Japanese eel mitochondrial genome sequenced, especially in the noncoding region.      

PCNA protein expression during spermatogenesis of the Japanese eel (Anguilla japonica)

Spermatogenesis can be initiated by a single injection of human chorionic gonadotropin (hCG) into the cultivated Japanese eel, which produces only spermatogonia in the testis. To isolate the genes responsible for regulating spermatogenesis, we performed a differential mRNA display using poly (A)+ RNA extracted from the testes at different time points after hCG injection. Among several cDNA clones, the expression of which was initiated before the onset of meiosis, one clone has high homology with the proliferating cell nuclear antigen (PCNA). In this study, we investigated the protein expression of eel PCNA and found for the first time in any species that two forms (32-kDa and 36-kDa) of PCNA are present in the testis. Although the 36-kDa form existed in both the testis and spleen, the 32-kDa form was specifically expressed in the testis. In contrast to the appearance of 36-kDa PCNA 1 day after the hCG treatment, the 32-kDa PCNA appeared only 9 days after the hCG treatment, at which time active spermatogonial proliferation occurred in the testis. Both the 32- and 36-kDa forms were recognized by antibodies raised against different epitopes of PCNA, and their N-terminal amino acid sequences were identical. The 36-kDa form, but not the 32-kDa form, was recognized by antibodies against phosphoamino acids. These results suggest that the two PCNA proteins are the same molecule with different chemical modifications, including phosphorylation. We discuss the roles of these two forms of PCNA in the spermatogenesis of the Japanese eel.     

Apolipoprotein A-II-mediated conformational changes of apolipoprotein A-I in discoidal high density lipoproteins

It is well accepted that HDL has the ability to reduce risks for several chronic diseases. To gain insights into the functional properties of HDL, it is critical to understand the HDL structure in detail. To understand interactions between the two major apolipoproteins (apos), apoA-I and apoA-II in HDL, we generated highly defined benchmark discoidal HDL particles. These particles were reconstituted using a physiologically relevant phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) incorporating two molecules of apoA-I and one homodimer of apoA-II per particle. We utilized two independent mass spectrometry techniques to study these particles. The techniques are both sensitive to protein conformation and interactions and are namely: 1) hydrogen deuterium exchange combined with mass spectrometry and 2) partial acetylation of lysine residues combined with MS. Comparison of mixed particles with apoA-I only particles of similar diameter revealed that the changes in apoA-I conformation in the presence of apoA-II are confined to apoA-I helices 3-4 and 7-9. We discuss these findings with respect to the relative reactivity of these two particle types toward a major plasma enzyme, lecithin:cholesterol acyltransferase responsible for the HDL maturation process.     

Characterization of microsatellite loci from the Japanese eel Anguilla japonica

The Japenese eel, Anguilla japonica, is generally assumed to be composed of a single population with wide distribution range, and some genetic studies using allozyme or mitochondrial DNA methods supported this population model. However, one genetic study suggested the existence of multiple populations in this species, and thus, more detailed studies on the population structure is needed. Here we characterized a total of 11 microsatellite markers of the Japanese eel. These will serve as powerful tools for detailed population study for the Japanese eel, though two of them showed the significant departure from the Hardy–Weinberg expectations.     

Pathological changes caused by cold-water stress in Japanese eel Anguilla japonica

In the present study, histopathological changes in Japanese eel Anguilla japonica subjected to 'cold-water stress' were examined. Eels were exposed to cold water (13 to 15 degrees C) and warmer water (25 degrees C) as controls. Fish held in warm water did not show any apparent changes. Although none of the eels exposed to cold water died, they displayed nephrotic changes such as cloudy swelling and hyaline droplet degeneration of the renal tubular epithelia. Fish with nephroses had low levels of serum chloride (12 to 23 mg l-1 in fish with hyaline droplet degeneration, 71 to 81 mg l-1 in fish with cloudy swelling) compared with the control fish (87 to 109 mg l-1). In electron microscopy, affected tubular cells had variously damaged mitochondria and formations of secondary lysosomes of variable sizes. Electron microscopy also revealed mitochondrial degeneration in hepatocytes and degenerated granules of neutrophils in the hematopoietic tissue. 'Cold-water stress' was effective in damaging Japanese eels below 15 degrees C.     

Antidipsogenic effects of eel bradykinins in the eel Anguilla japonica

A peptide with bradykinin (BK)-like immunoreactivity was isolated from an incubate of heat-denatured eel plasma with porcine pancreatic kallikrein. The purified peptide had the following amino acid sequence: Arg-Arg-Pro-Pro-Gly-Ser-Trp-Pro-Leu-Arg. This decapeptide, named eel [Arg(0)]BK, was identical to two previously identified BK homologs from cod and trout. High conservation of the BK sequence among distant teleost species suggests an important function in this vertebrate group. Bolus intra-arterial injections of eel [Arg(0)]BK, BK, and [Arg(0)]-des-Arg(9)-BK (1-10 nmol/kg) caused significant (P < 0.05) inhibition of drinking in seawater-adapted eels. The potency of the inhibition was ranked in the following order: [Arg(0)]BK > [Arg(0)]-des-Arg(9)-BK = BK. The BK peptides also produced an immediate, transient increase followed by a sustained increase in arterial blood pressure and an initial decrease followed by an increase in heart rate. Strong tachyphylaxis occurred for the cardiovascular effect but not for the antidipsogenic effect. The order of the potency of the cardiovascular actions, [Arg(0)]BK > BK > [Arg(0)]-des-Arg(9)-BK, was different from that of the antidipsogenic action. Slow infusions of eel [Arg(0)]BK in the dose range 1-1,000 pmol x kg(-1) x min(-1) produced concentration-dependent inhibition of drinking without changes in arterial pressure, plasma osmolality, and hematocrit. At the infusion rate of >100 pmol x kg(-1) x min(-1), plasma concentrations of angiotensin II, a potent dipsogenic hormone in eels, increased, suggesting an interaction of the kallikrein-kinin and renin-angiotensin systems. In mammals, BK is dipsogenic and vasodepressor, so that our data demonstrate opposite effects on fluid and cardiovascular regulation of BK in the eel and suggest a new physiological role for the kallikrein-kinin system in teleost fish.     

A novel progestogen receptor subtype in the Japanese eel, Anguilla japonica.

A cDNA encoding a second type of a progestogen receptor (ePR2) was isolated from the same library as we had previously cloned a functional PR (ePR1) in eel testis. The amino acid sequence of the ePR2 shows low homology with ePR1 (34%), but both PRs showed progestogen-dependent transactivation in transfection experiment. Tissue distribution of ePR2 mRNA was clearly different from that of ePR1. Protein interaction between two PRs was demonstrated in vitro by a glutathione S-transferase pull-down assay. These results indicate that ePR2 is also a functional PR. This is the first isolation of two different functional PR molecules from a vertebrate.     

"Blood-contacting neurons" in the brain of the Japanese eel Anguilla japonica

To discriminate "blood-contacting neurons" within the brain of the eel, Evans blue (EB) was injected intraperitoneally. After five days, six brain areas were externally stained blue with the dye; the saccus dorsalis (SD), the epiphysis (E), the area postrema (AP), the posterior part of the magnocellular preoptic nucleus (PM), the pituitary (Pit), and the saccus vasculosus (SV). Among the EB-positive area, some cells in the PM, the anterior tuberal nucleus (NAT) and the AP were discriminated as the "blood-contacting neurons" histologically, whereas EB-positive neurons were not detected in the SD, the E, the Pit and the SV regions. In the PM, most EB-positive neurons (90 %) were immunoreactive to vasotocin (AVT) antibody, indicating that these neurons are vasotocinergic. The remaining EB-positive neurons (10 %) were not immunoreactive to ANG II and tyrosine hydroxylase (TH) antibodies. Although some neurons in the PM were immunoreactive to ANG II antibody, they were EB-negative. In contrast, almost all EB-positive neurons in the AP showed TH-like immunoreactivity (-lir), indicating that these neurons utilize catecholamine(s) as a neurotransmitter. The EB-positive neurons in the NAT were not immunoreactive to AVT, ANG II and TH antibodies, whereas some neurons without EB-staining showed ANG II-lir. Possible roles of these neurons in regulating drinking behavior in eels are discussed.     

Evaluation of downward movements of Japanese eel Anguilla japonica inhabiting brackish water areas

This study evaluated the size and age distributions and otolith microchemistry of the Japanese eel Anguilla japonica in freshwater and brackish water areas in the Aki and Tsuchikawa rivers for 1 year, and in brackish water areas in the Asahi River for 3 years to understand the movements of Japanese eels between continental habitats of different salinity after recruitment (n = 759). For all three rivers, the total length (L_T) and age distributions were consistent; yellow eels captured in the upper brackish water (Aki River: 353.5 ± 77.4 mm and 3.0 ± 0.8 years; Tsuchikawa River: 287.7 ± 87.3 mm and 3.7 ± 1.3 years; Asahi River: 418.2 ± 112.1 mm and 4.2 ± 1.7 years) were smaller and younger than not only those in the fresh water of the two rivers but also those in the lowest brackish water sampling areas (Aki River: 436.0 ± 71.6 mm and 3.8 ± 1.1 years; Tsuchikawa River: 370.9 ± 121.7 mm and 4.9 ± 2.3 years; Asahi River: 558.5 ± 85.9 mm and 5.7 ± 1.7 years). In the Asahi River, these tendencies were found throughout the 3 years. Otolith analysis indicated that the majority of the eels captured in the lowest brackish water areas had moved down from upstream. These results suggest that Japanese eels inhabiting saline water generally move from the upper estuary as they grow. The upper estuary can be an important area for the management of this species because these eels spend their early continental growth life there.     

Follicle-stimulating hormone induces spermatogenesis mediated by androgen production in Japanese eel, Anguilla japonica

Follicle-stimulating hormone (FSH) plays important roles in spermatogenesis. However, the biologic activity of FSH can vary in different vertebrate classes, and the definitive function of FSH has not been established. In this study, we investigated the functions of FSH on spermatogenesis using an in vitro culture system for Japanese eel testis. The eel Fsh receptor was expressed in testis tissue during the whole process of spermatogenesis, mainly by Leydig cells that produce steroid hormones and by Sertoli cells surrounding type A spermatogonia and early type B spermatogonia. In an in vitro organ culture, recombinant eel Fsh (r-eFsh) induced complete spermatogenesis from the proliferation of spermatogonia to spermiogenesis during 36 days of culture; also, spermatozoa were observed in the testicular fragments. Spermatogenesis induced by r-eFsh was inhibited by trilostane, a specific inhibitor of 3beta-hydroxysteroid dehydrogenase. However, trilostane did not inhibit spermatogenesis induced by 11-ketotestosterone. These results clearly show that the main function of FSH in eel is to induce spermatogenesis via stimulating androgen production.     

Location,size and age at onset of metamorphosis in the Japanese eel Anguilla japonica

This study clarifies the location, size and age at the onset of metamorphosis in Japanese eels Anguilla japonica through oceanic surveys, rearing experiments and analyses of the morphology and otoliths of leptocephali and glass eels. Twenty‐eight metamorphosing leptocephali were collected in the mesoscale eddy region to the east of Taiwan during research expeditions in 2004. Rearing experiments showed that the total length (L_T) of leptocephali decreased by an average of 12·5% during metamorphosis and 13·9% during the 2–12 h after death. Thus, the mean back‐calculated L_T at the onset of metamorphosis for 630 glass eels from Taiwan and Japan was estimated at 67·8 ± 2·7 mm (mean ± S.D.). The estimated mean ante‐mortem size of the fully grown pre‐metamorphic leptocephali collected in 2004 was 64·6 ± 3·4 mm, which was consistent with the L_T estimate for glass eels. Otolith analysis showed that the mean age at the onset of metamorphosis was 137 ± 15 days and indicated that Japanese eels may have a recruitment route through the mesoscale eddies to the east of Taiwan in addition to the direct transfer route from the North Equatorial Current to the Kuroshio Current.     

Serum estradiol-17beta and testosterone levels during silvering in wild Japanese eel Anguilla japonica

To understand the changes of serum levels of sex steroids in the wild Japanese eel Anguilla japonica during silvering process, eels collected from the Kaoping River of Taiwan from August 2000 through June 2001 were examined. The maturational stages of female eels before and during silvering were divided into four stages: juvenile, sub-adult, pre-silver and silver stages based on skin coloration and oocyte diameter. Male eels were investigated only in the silver stage. Radioimmunoassays were employed to measure serum levels of estradiol-17β (E₂) and testosterone (T). The mean liver mass of the female eels increased significantly during silvering, but the mean hepatosomatic index remained constant. In contrast, mean ovarian mass and gonadosomatic index increased significantly during silvering. Serum concentrations of E₂ in females increased significantly during silvering (P<0.05), while E₂ was undetectable in silver males. The mean serum T concentrations increased significantly in females (P<0.05) during silvering, with lowest mean values in the juvenile stage and highest mean value in the silver stage. The mean serum T level in the silver males was significantly lower than in silver females (P<0.05). In conclusion, both serum E₂ and T concentrations increased with ovarian development of wild Japanese eels during silvering, while serum E₂ was undetectable in the silver male eels. The findings support the idea that androgen, but not estrogen, plays a major role in silvering process of the eels in both sexes.     

Daily growth increments in the larval otolith of the Japanese eel,Anguilla japonica

Early formation of otolith was studied on artificially hatched larvae of the Japanese eel,Anguilla japonica. Newly hatched larvae had a pair of sagittae which were flat and subelliptical with 8.3 μm in mean diameter. The diameter of the sagitta increased linearly with age. No growth increments were observed in the sagitta at hatching, while larvae which were 2, 4 and 6 days old had on average 2.1, 3.6 and 6.0 increments, respectively. The number of the increments (Y) and the age in days after hatching (X) showed a close linear relationship (Y=0.96X+ 0.06, r = 0.913, n = 40), suggesting daily deposition of sagittal increments. In 95 % of the field-caught elvers of this species, a distinct dark ring (check) with the diameter of 6–12 μm was found around the nucleus of the sagitta. This seems to be a “hatch check” deposited at hatching, since its diameter roughly agreed with that of the sagitta in the newly-hatched larvae. Possibly, the number of the increments outside the hatch check represents the age of the fish in days.     

Recruitment mechanism of the eel, Anguilla japonica, to the Japanese coast

A total of 149 Anguilla japonica elvers collected at 10 locations along the Japanese coast were aged according to otolith daily increments. The interrelationships among age, birth date, body size, pigmentation stage, sampling location and the timing of recruitment were examined in order to determine the recruitment mechanism of elvers to coastal waters.
First catches of eels were earlier in the locations at lower latitudes. Age at recruitment was roughly constant, 218 ± 29 (mean ± s.d.) days old, disregarding the localities, but a weak positive correlation was obtained between age and sampling date or timing of recruitment. Body length at recruitment was also constant, 56.3 ± 2.3 mm s.l., and showed no significant correlation with either locality or recruitment timing. Estimated birth date ranged from April to November, the mean ± s.d. being 22 July 1982 ± 42 days, suggesting a peak season spawning in summer. Birth date was closely related with both the latitude of location and sampling time. Pigmentation developed more at lower latitude. Recruitment mechanism of the Japanese eel was summarized as follows: the earlier-born individual recruits earlier at lower latitude and at younger age, but at a constant body size.