Effects of temperature on vitellogenesis in Japanese eel Anguilla japonica

ABSTRACT:   Cultured immature female Japanese eels acclimated to sea water at either 10 or 20°C were treated weekly with salmon gonadotropin fraction (sGTH) in order to investigate the effects of water temperature on artificial induction of ovarian maturation. In eels maintained at 20°C, ovulation was induced in 11 of 18 fish during the experimental period of 13 weeks, whereas at 10°C all fish showed a low gonadosomatic index (GSI) at the end of the experiment. Plasma vitellogenin levels were higher in eels kept at 20°C than in eels at 10°C throughout the experiment. However, no significant differences were observed in the plasma testosterone and estradiol-17β levels between groups. Eels pretreated with sGTHs at 10°C for 13 weeks were separated to two different temperature (10 and 20°C) groups, and received the same weekly sGTH injections. In eels transferred to 20°C, plasma vitellogenin levels, GSI and oocyte diameter were increased, but these values were maintained at low levels in eels that remained at 10°C. These results clearly indicate that water temperature is an important factor regulating vitellogenesis in the Japanese eel.     

Development of an emulsion prepared with lipophilized gelatin and its application for hormone administration in the Japanese eel Anguilla japonica

A new water-in-oil-in-water (W/O/W) type emulsion using lipophilized gelatin (LG) and cotton-seed oil was developed for a novel method of hormone administration in fishes. LG was prepared by attaching palmitic anhydride to gelatin. The best conditions for preparing stable emulsions were determined where the final concentrations of LG in the emulsion and the volume ratio of LG solution to cotton-seed oil were 2% and 1:2, respectively. Hormone releasing properties of LG emulsion were compared with a water-in-oil (W/O) type emulsion prepared with Freund's incomplete adjuvant (FIA) and saline solution by monitoring plasma profiles of salmon gonadotropin (sGtH) II in the Japanese eel. sGtH II is gradually released from the LG emulsion. LG emulsion had hormone-releasing properties different from those of the saline solution and the FIA emulsion. Immature female Japanese eel (BW, 566 to 1017 g) received a weekly intramuscular injections (total of 10 injections) of the LG emulsion, the FIA emulsion or the saline solution, each of which contained sGtH fraction. The LG emulsion was found to be more effective in inducing ovarian maturation. In mature eel, final maturation and ovulation could be induced by simultaneous administration of sGtH fraction and 17-hydroxyprogesterone using LG W/O/W emulsion.     

Changes in serum vitellogenin levels and immunohistochemical localization of vitellogenin in hepatic cells during ovarian development in the Japanese eel

ABSTRACT: The present study was designed to quantify changes in serum vitellogenin (VTG) levels and to localize VTG in hepatic cells immunohistochemically during artificial maturation of the Japanese eel Anguilla japonica . Hormone treatment resulted in a gradual rise in serum VTG levels, in which the highest values were attained at the migratory nucleus stage. Hepatocytes responded to hormone treatment by increased immunoreactivity; both staining area and intensity were enhanced with ongoing gonadal development. Moreover, cytoplasmic vacuoles increased in number during artificial maturation, occupying large areas inside hepatocytes. The unusual appearance of hepatocytes may reflect the effect of artificial maturation.     

日本鳗鲡排卵的人工诱导

对日本鳗鲡(Anguillajaponica)卵巢发育的时间与催产率的关系进行研究,对卵母细胞在体内胚泡破裂(GVBD)的发生过程、以及用17α-OHP和17α,20β-DHP诱导日本鳗鲡排卵的方式、时机和催产效果进行观察,结果表明,卵巢发育时间越长,催产率越低。造成催产率低的原因主要是由于日本鳗鲡在卵母细胞成熟后期,雌鳗个体对催产药物敏感性差异较大,催产时机难以掌握。通过采用适合日本鳗鲡卵母细胞的透明液观察GVBD的发生过程,并根据每个个体GVBD的发生速度,制订适宜的催产方案基本解决了上述难题。本实验采用CP、HCG、17α-OHP或17α,20β-DHP混合催产,使其平均催产率由35 4%分别提高到91 9%和93 0%。17α-OHP和17α,20β-DHP的催产剂量为5mg/(500g)和10mg/(500g)。在(21±0 5)℃水温条件下,催产效应时间分别为15～18h和13～16h。     

Genetic variability of the Japanese eel Anguilla japonica (Temminck & Schlegel) related to latitude

Abstract— Isozyme genotypes of 400 glass eels recruiting to 4 localities along the east Asian coast, stretching from Taiwan to the Yalu River of northeastern China, were studied using starch gel electrophoresis. Geographic cline was found to exist in two loci: NADP-isocitrate dehygenase-1 and 6-phosphogluconate dehydrogenase. Frequencies of the most common allele of these two loci increased from south to north. In the latitudinal range of 25°N to 40°N, the magnitude of difference of IDH100 and PGD100 was 13% and 9% respectively. However, deviations from Hardy-Weinberg equilibrium were found to be insignificant in both loci at three of the four localities. The cline was, therefore, unlikely to have resulted from selection. Migration time-lag from different parts of the continent to the spawning ground in the western Pacific was suggested to be a possible reason for the formation of the cline.     

Induction of spermatogenesis in Japanese eel by recombinant goldfish gonadotropins

We previously demonstrated the biological activities of single-chain recombinant gonadotropins (scGTHs) of goldfish Carrassius auratus follicle-stimulating hormone (scFSH) and luteinizing hormone (scLH), produced by a baculovirus–silkworm larvae system, by using in vivo bioassays with some fishes including Japanese eel Anguilla japonica. Among the bioassays, we succeeded in induction of spermatogenesis of sexually immature male Japanese eels by both scFSH and scLH, especially resulting in the occurrence of spermatozoa in scLH-administered males. However, those recombinant hormones did not induce enlargement of testes. In order to further confirm the potency of recombinant GTHs for use in aquaculture species, we administered scFSH and scLH to males of Japanese eel at higher dosage and frequency (eight times with 2–5 days interval) than those of the previous study (five or six times with 7 days intervals), including combination of scFSH and scLH administration (scFSH–scLH). Gonadosomatic indices (GSI) of scLH- and scFSH–scLH-administered males were larger than those of initial control males and of control males that were injected with saline. Enlargement of testes was also confirmed by measurement of testicular lobe size in scFSH-, scLH-, and scFSH–scLH-administered males. By histological observation, occurrence of spermatozoa was confirmed in scLH- and scFSH–scLH-administered eels. Although milt production was not induced, higher dosage and frequency of scGTH administration was effective in promoting testicular development of immature eels. Thus, single-chain fish GTHs produced by the baculovirus–silkworm larvae system could be a useful tool for promotion of gonadal maturation in aquaculture fishes.     

Cardiovascular,respiratory, and blood adjustments to hypoxia in the Japanese eel,Anguilla japonica

The cardioventilatory performance of the Japanese eel,Anguilla japonica, was evaluated during acute exposure to hypoxia. The eel became an oxygen conformer as ambient PO₂ fell below the critical value of 110 mmHg. Although arterial and venous PO₂ also fell progressively, the arterial O₂ content remained constant down to an ambient PO₂ of about 60 mmHg. Arterial blood O₂ saturation was maintained at 85% even at 40 mmHg. The increase in the supply of O₂ to the animal during hypoxia was due to a combination of adaptive adjustments: (1) an increase in ventilation: perfusion ratio brought about mainly be bradycardia; (2) an increase in respiratory exchange surface area which was manifested as an increase in branchial blood transit time and quantified as a rise in transfer factor, water-blood overlap coefficient and utilization (%); (3) an increase in blood O₂ affinity and capacitance coefficient as a result of respiratory alkalosis and Bohr-Root shift and a decrease in haemoglobin allosteric modulator (GTP, ATP) concentrations in the RBC. These factors together helped to increase the efficiency of O₂ transfer across the gills.     

Investigation into the possible role of androgens in the induction of hepatic vitellogenesis in the European eel:in vivo andin vitro studies

Changes in the levels of plasma vitellogenin (Vg), estradiol (E₂) and testosterone (T) were examined following gonadal development induced by carp gonadotropin treatment (cGTH) of freshwater female yellow and silver eels (Anguilla anguilla L.). The animals received injections of cGTH (250 μg kg⁻¹ body weight) or saline vehicle three times a week, for 6 to 8 weeks. No effect of vehicle was observed. Steroidogenic activity of the ovary was stimulated by cGTH treatment as shown by the increase in circulating steroid levels in both stages. However, the responses of T, E₂ and Vg differed according to the stage of development of eels. At the yellow stage, the initial steroid plasma levels were undetectable (< 0.01 ng ml⁻¹) before treatment and ovarian steroidogenic activity was slightly stimulated following cGTH treatment; steroid levels reached their highest values after 3 weeks and 6 weeks of treatment for E₂ (0.62 ± 0.13 ng ml⁻¹ and T (0.33 ± 0.30 ng ml⁻¹), respectively. The cGTH treatment slightly increased plasma Vg levels (0.2–0.7 μg ml⁻¹ during the experiment compared with the initial values of the group. At the silver stage, the initial steroid levels were detectable (0.7 ng ml⁻¹ for E₂ and 0.1 ng ml⁻¹ for T); cGTH treatment did not significantly increase plasma E₂ level which remained at initial levels. Nevertheless, plasma T levels dramatically increased from 0.1 to 3 ng ml⁻¹ and peaked after 1 or 2 weeks of cGTH treatment; a rapid increase in plasma Vg levels occurred, reaching its highest value at 5 mg ml⁻¹ after 3 weeks of treatment. Thus, the steroid kinetic profiles in relation to the appearance of Vg in the plasma following cGTH treatment was closely related to androgen levels and there was a strong vitellogenic response induced by chronic cGTH treatment. In order to test if androgens could be implicated in the vitellogenic response, we evaluated the potencies of various androgens (testosterone and 5α-androstane-3β,17β-diol)in vivo andin vitro, associated with E₂ to induce the production of Vg.In vitro experiments showed that Vg synthesis was induced by high doses (10⁻⁶ to 10⁻⁵ M) of androgen in the eel. Tamoxifen totally inhibited the action of androgens suggesting that androgens were acting through binding to the E₂ receptor.In vivo, androgens given alone at 50 μg kg⁻¹ 3 times a week for 1 months had no significant effect on plasma Vg levels. In addition, E₂-androgen cotreatment showed that the presence of androgen did not modify the vitellogenic response induced by E₂.     

Artificial induction of maturation and fertilization in the Japanese eel, Anguilla japonica

Repeated injections of salmon pituitary extract (20 mg per fish per week) induced vitellogenesis in feminized, cultivated Japanese eels (Anguilla japonica). Oocytes were attained at the migratory nucleus stage after 11 or 12 injections. Addition of 17,20-dihydroxy-4-pregnen-3-one (DHP) into the incubation medium induced germinal vesicle breakdown (GVBD) in the oocytes at the migratory nucleus stage. An injection of DHP (2 µg g^-1 BW), given 24h after an injection of salmon pituitary extract (20 mg fish^-1), succeeded in inducing maturation and ovulation in females which contained occytes at the migratory nucleus stage. Most fish ovulated 15–18h following the DHP injection. Eggs that were ovulated within 15h after the DHP injection showed high fertility and hatchability, but eggs ovulated 18 or 21h after the DHP injection, showed considerably lower fertility and hatchability. A delay between ovulation and stripping of the eggs rapidly decreased both the fertility and hatchability within 6–9h after ovulation, indicating that artificial fertilization must be carried out immediately after ovulation. Repeated injections of human chorionic gonadotropin (hCG) at a concentration of 1 IU g^-1 BW week^-1 induced spermatogenesis, spermiation, and the acquisition of potential for sperm motility in cultivated males. Most males spermiated after the fifth or sixth injection of hCG, and the milt weight gradually increased and remained constant (1–2 g) from the 11th to 31th injection. Sperm motility peaked 24h after each weekly injection, and decreased from the 3^rd day after the injection. Potassium ions are an essential constituent for the maintenance of motility in the eel spermatozoa. Artificial seminal plasma containing 15.2 mM KCl is applicable as a milt diluent. Using these techniques developed for female and male eels, we have succeeded in obtaining many fertilized eggs from cultivated eels.     

Immunohistochemical changes in production of pituitary hormones during artificial maturation of female Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis>

ABSTRACT:   Specific antibodies against follicle-stimulating hormone β subunit (FSHβ), prolactin (PRL), and somatolactin (SL) of the Japanese eel Anguilla japonica were produced. These antibodies, as well as antibodies against luteinizing hormone β subunit (LHβ) and growth hormone (GH) produced previously, were used to examine changes in the production of pituitary hormones in female eels during maturation induced by salmon pituitary homogenate (SPH) injection. Immunohistochemical observations showed a decrease in FSH production after SPH injection, suggesting that SPH inhibits FSH production. In contrast, LH production increased markedly with maturation. The number of GH producing cells decreased gradually during maturation, possibly because of inhibition by exogenous GH present in the SPH and/or endogenous insulin-like growth factor-I produced by the stimulation of salmon GH. Although changes in the number of PRL producing cells with maturation were not evident, the number of SL producing cells showed a peak at the late vitellogenic stages, and thereafter decreased to the migratory nucleus stage. These results suggest that GH and SL are involved in sexual maturation in SPH injected eels, as in other fishes.     

Role of prostaglandin in the control of ovulation in the Japaneseeel Anguilla japonica

ABSTRACT:   The in vitro effects of 17,20β-dihydroxy-4-pregnen-3-one(DHP) and prostaglandins (PGE₁, PGE₂, PGF_1α,PGF_2α) on ovulation in the Japanese eel Anguillajaponica were examined. Oocytes with follicle layers at themigratory nucleus stage (approximately 850–900 µmdiameter) were removed using a polyethylene cannula from artificiallymatured fish. At concentrations of 10 and 100 ng/mL,DHP was found to induce both germinal vesicle breakdown and ovulation.The prostaglandins, except for PGE₁, effectively inducedovulation of previously matured oocytes by DHP treatment in vitro .Prostaglandin F_2α was the most effective. Asignificant increase in ovulation rate was observed even at a concentrationof 0.01 µg/mL PGF_2α.Indomethacin blocked the in vitro ovulation induced by DHPand addition of PGF_2α reversed indomethacin-blockedovulation. Actinomycin D and cycloheximide blocked DHP-induced ovulationand PGF_2α reversed the effects of both inhibitors. Theseresults indicate that DHP induces ovulation through endogenous prostaglandinsynthesis in the follicle layers of the Japanese eel.     

Fine structure and differentiation of the alimentary canal in captive-bred Japanese eel Anguilla japonica preleptocephali

ABSTRACT:   The fine structure of the alimentary canal in preleptocephali produced by artificially matured Japanese eel was examined. At 1 day posthatch (dph), the alimentary canal was found only above the dorsal side of the yolk mass, and the epithelium was composed of a single layer of epithelial cells. By 5 dph, the alimentary canal was divided into three segments based on the structure of the epithelial cells: foregut, midgut and hindgut, corresponding to the future esophagus, intestine and rectum, respectively. After 7 dph, the epithelium in the foregut was surrounded by a circular muscle layer, suggesting a role in the transportation of food materials. The epithelial cells of the midgut exhibited well-developed membranous structures, which are deduced to be invaginations of the cytoplasmic membrane. Pinocytotic invaginations and vacuoles were observed in the epithelial cells of the hindgut; this observation suggests that this region is involved in the uptake of food. Significant changes in morphological features of the epithelial cells in each segment were observed until 7 dph; however, these were not evident between 7 dph and 13 dph. Consequently, the differentiation of the alimentary canal was completed by 7 dph, and preleptocephalus had developed the ability to absorb food by 7 dph.     

Insulin-like growth factor I of Japanese eel, Anguilla japonica: cDNA cloning, tissue distribution, and expression after treatment with growth hormone and seawater acclimation

In an attempt to understand growth regulation in the Japanese eel, Anguilla japonica, we cloned insulin-like growth factor-I (IGF-I) cDNAs and examined their mRNA expression in several tissues. Two eel IGF-I (eIGF-I) cDNAs encoding preprohormones, eIGF-I-Ea1and eIGF-I-Ea2, were cloned from the liver by polymerase chain reaction (PCR). The preproIGF-Is were identical in signal peptide and mature IGF-I, but different in the E domain—eIGF-I-Ea2 mRNA was 36 bp longer than eIGF-I-Ea1 mRNA. Eel IGF-I was 83–94% identical with that of teleosts, 71% identical with that of dogfish, 87% identical with that of bullfrog and chicken, and 83% identical with that of humans. In both males and females the highest eIGF-I-Ea1 mRNA levels were observed in the liver, with detectable levels also found in the gills, heart, stomach, spleen, kidney, intestine, swim-bladder, muscle, and gonads. eIGF-I-Ea1 mRNA levels in the liver were higher in females than in males whereas in the intestine they were lower than in males. eIGF-I-Ea2 mRNA was detected in all the tissues examined and at similar levels in males and females. In this experiment higher eIGF-I-Ea1 mRNA levels were observed in the liver of larger glass eels than in those of smaller fish. eIGF-I-Ea2 mRNA levels were also higher in larger eels, although they were lower than IGF-I-Ea1 mRNA levels. Both eIGF-I mRNA levels in liver were positively correlated with the body size of the␣glass eels. Intraperitoneal injection of recombinant eel GH (reGH), 0.25 μg g⁻¹ body weight, into glass eels resulted in a significant increase in both eIGF-I mRNAs in the liver 1 day after injection compared with control fish, but no elevation was observed 2 days after injection. Incubation of liver slices with reGH at concentrations of 10, 100, and 1,000 ng mL⁻¹ for 24 h resulted in a significant concentration-dependent increase in the levels of both eIGF-I mRNAs. Higher levels of eIGF-I-Ea1 and Ea2 mRNA were observed in the gills ofseawater-reared eels than in those of freshwater-reared fish, but no differenceswere observed in the whole kidney. These results suggest that IGF-I is involved in the regulation of somatic growth and also in adaptation of the Japanese eel to seawater.