The expression profiles of cyp19a1, sf-1, esrs and gths in the brain-pituitary during gonadal sex differentiation in juvenile Japanese eels.

Eels are gonochoristic species whose gonadal differentiation initiates at the yellow eel stage and is influenced by environmental factors. We revealed some sex-related genes were sex dimorphically expressed in gonads during gonadal sex differentiation of Japanese eel (Anguilla japonica); however, the expression of sex-related genes in the brain-pituitary during gonadal sex differentiation in eels is still unclear. This study aimed to investigate the sex-related gene expressions in the brain-pituitary and tried to clarify their roles in the brain and gonads during gonadal sex differentiation. Based on our previous histological study, the control eels developed as males, and estradiol-17ß (E2) was used for feminization. Our results showed that during testicular differentiation, the brain cyp19a1 transcripts and aromatase proteins were increased significantly; moreover, the cyp19a1, sf-1, foxl2s, and esrs (except gperb) transcripts in the midbrain/pituitary also were increased significantly. Forebrain gnrh1 transcripts increased slightly during gonadal differentiation of both sexes, but the gnrhr1b and gnrhr2 transcripts in the midbrain/pituitary were stable during gonadal differentiation. The expression levels of gths and gh in the midbrain/pituitary were significantly increased during testicular differentiation and were much higher in males than in E2-feminized females. These results implied that endogenous estrogens might play essential roles in the brain/pituitary during testicular differentiation, sf-1, foxl2s, and esrs may have roles in cyp19a1 regulation in the midbrain/pituitary of Japanese eels. For the GnRH-GTH axis, gths, especially fshb, may be regulated by esrs and involved in regulating testicular differentiation and development in Japanese eels.

Induction of oocyte development in previtellogenic eel, Anguilla australis.

The role of gonadotropins during early ovarian development in fish remains little understood. Concentrations of gonadotropins were therefore experimentally elevated in vivo by administration of recombinant follicle-stimulating hormone (rec-Fsh) or human chorionic gonadotropin (hCG) and the effects on ovarian morphology, sex steroid levels and mRNA levels of genes expressed in pituitary and ovary examined. Hormones were injected thrice at weekly intervals in different doses (20, 100 or 500 µg/kg BW for rec-Fsh and 20, 100 or 500 IU/kg BW for hCG). All treatments, especially at the highest doses of either rec-Fsh or hCG, induced ovarian development, reflected in increased oocyte size and lipid uptake. Both gonadotropins up-regulated follicle-stimulating hormone receptor (fshr) mRNA levels and plasma levels of estradiol-17ß (E2). Exogenous gonadotropins largely decreased the expression of follicle-stimulating hormone ß-subunit (fshb) and had little effect on those of luteinizing hormone ß-subunit (lhb) in the pituitary. It is proposed that the effects of hCG on ovarian development in previtellogenic eels could be indirect as a significant increase in plasma levels of 11-ketotestosterone (11-KT) was found in eels treated with hCG. Using rec-Fsh and hCG has potential for inducing puberty in eels in captivity, and indeed, in teleost fish at large.

Does silvering or 11-ketotestosterone affect osmoregulatory ability in the New Zealand short-finned eel (Anguilla australis)?

Silvering has been associated with advancing osmoregulatory ability. Given the demonstrated role of 11-ketotestosterone (11KT) in mediating many of the silvering-related changes, we investigated the role of 11KT in driving this advanced osmoregulatory ability in the New Zealand short-finned eel (Anguilla australis). Yellow (non-migratory) eels with or without 11KT implants and blank-implanted silver (migratory) eels, either held in freshwater or subjected to seawater challenge, were sampled to determine serum [Na+] and [Cl-], pituitary prolactin mRNA levels, gill Na+/K+-ATPase activity and gill mRNA levels for Na+/K+-ATPase-α1 subunit and for Na+/K+/2Cl- co-transporter-1α-subunit. Developmental stage and 11KT treatment advanced the eels' osmoregulatory ability. Thus, serum [Na+] and [Cl-] were affected by developmental stage and 11KT treatment upon seawater challenge. However, seawater challenge, not 11KT treatment or developmental stage, produced the strongest and the most consistent effects on A. australis osmoregulatory processes, inducing significant effects in all the relevant parameters we measured. In light of our results and in view of the eel's marine ancestry, we contend that A. australis, or freshwater eels in general, are highly tolerant and able to adapt quickly to changing salinities even at the yellow stage, which may preclude a critical need for an advanced osmoregulatory ability at silvering.

Androstenedione and 17α-methyltestosterone induce early ovary development of Anguilla japonica.

Endocrine effects as 11-ketotestosterone (11-KT), an unaromatizable androgen, regulating the follicles growth in the previtellogenic stage of eel reproduction have been widely elucidated. However, the influence of aromatizable androgens on the brain-pituitary-gonad axis during oogenesis in A. japonica has not been clearly elaborated. In the study, androstenedione (AD) and 17α-methyltestosterone (MT) were employed together to induce ovary development of seven-year-old female Anguilla japonica through feeding or exposure in the migration season. After female A. japonica had been fed with commercial diet containing 5 mg AD and MT kg d-1 body weight respectively for 45 d in fresh water (Trial I), the development of oocytes still remained at the oil droplet stage, but the GSI and follicle diameter increased significantly. The serum 11-KT level and expression of liver vitellogenin mRNA were significantly elevated. After female fish had been exposed to seawater containing 50 µg L-1 AD and MT respectively for 45 d (Trial II), the ovaries of A. japonica almost reached midvitellogenic stage and the GSI and follicle diameter increased significantly. Yolk granular layer was observed in the peripheral ooplasm. The serum 11-KT level maintained consistently low, and the serum E2 level declined significantly to a relatively low level. The expression levels of ovarian arα and cyp19a1, brain (with pituitary together) mGnRH and lhß increased significantly. The results showed that A. japonica in Trial II appeared a higher ovarian development than those in Trial I. These findings indicated that AD and MT increased the oil droplet and enlarged follicle diameter in previtellogenic stage, while the vitellogenesis and gonadotropin release did not occur in Trial I. In Trial II, AD and MT promoted vitellogenesis by stimulating the ovary expression of arα and by up-regulating brain mGnRH and pituitary lhß expression.

Interactive effects of dietary composition and hormonal treatment on reproductive development of cultured female European eel, Anguilla anguilla.

Farmed female eels were fed two experimental diets with similar proximate composition but different n-3 polyunsaturated fatty acid (PUFA) levels. Both diets had similar levels of arachidonic acid (ARA), while levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in one diet were approximately 4.5 and 2.6 times higher compared to the other diet, respectively. After the feeding period, each diet group was divided into two and each half received one of two hormonal treatments using salmon pituitary extract (SPE) for 13 weeks: i) a constant hormone dose of 18.75mg SPE/kg initial body weight (BW) and ii) a variable hormone dosage that increased from 12.5mg SPE/kg initial BW to 25mg SPE/kg initial BW. Results showed a significant interaction between diets and hormonal treatments on gonadosomatic index (GSI), indicating that the effect of broodstock diets on ovarian development depends on both nutritional status and hormonal regime. Females fed with higher levels of n-3 series PUFAs and stimulated with the constant hormonal treatment reached higher GSIs than those receiving the variable hormonal treatment. However, when females were fed lower levels of n-3 series PUFAs there was no difference in the effect of hormonal treatments on GSI. We also found that, independent of hormonal treatment, the diet with higher levels of n-3 series PUFAs led to the most advanced stages of oocyte development, such as germinal vesicle migration. Concentration of sex steroids (E2, T, and 11-KT) in the plasma did not differ between diets and hormonal treatments, but was significantly correlated with ovarian developmental stage. In conclusion, increasing dietary levels of n-3 PUFAs seemed to promote oocyte growth, leading to a more rapid progression of ovarian development in European eel subjected to hormonal treatment.

Characterization and expression of cDNAs encoding P450c17-II (cyp17a2) in Japanese eel during induced ovarian development.

Estradiol-17ß (E2) and maturation-inducing hormone (MIH) are two steroid hormones produced in the teleost ovary that are required for vitellogenic growth and final oocyte maturation and ovulation. During this transition, the main steroid hormone produced in the ovary shifts from estrogens to progestogens. In the commercially important Japanese eel (Anguilla japonica), the MIH 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) is generated from its precursor by P450c17, which has both 17α-hydroxylase and C17-20 lyase activities. In order to elucidate the regulatory mechanism underlying the steroidogenic shift from E2 to DHP and the mechanistic basis for the failure of this shift in artificially matured eels, the cDNA for cyp17a2-which encodes P450c17-II-was isolated from the ovary of wild, mature Japanese eel and characterized, and the expression patterns of cyp17a1 and cyp17a2 during induced ovarian development were investigated in cultured eel ovaries. Five cDNAs (types I-V) encoding P450c17-II were identified that had minor sequence variations. HEK293T cells transfected with all but type II P450c17-II converted exogenous progesterone to 17α-hydroxyprogesterone (17α-P), providing evidence for 17α-hydroxylase activity; however, a failure to convert 17α-P to androstenedione indicated that C17-20 lyase activity was absent. Cyp17a2 mRNA was expressed mainly in the head kidney, ovary, and testis, and quantitative PCR analysis demonstrated that expression in the ovary increased during induced vitellogenesis and oocyte maturation/ovulation. In contrast, P450c17-I showed both 17α-hydroxylase and C17-20 lyase activities, and cyp17a1 expression increased until the mid-vitellogenic stage and remained high thereafter. Considering the high level of cyp17a2 transcript in the eel ovary at the migratory nucleus stage together with our previous report demonstrating that eel ovaries have strong 17α-P-to-DHP conversion activity, the failure of artificially maturing eels to produce the maturation-inducing DHP may be explained by a deficiency in 17α-P production due to the persistence of cyp17a1 expression after the completion of vitellogenesis.

Abnormal ovarian DNA methylation programming during gonad maturation in wild contaminated fish.

There is increasing evidence that pollutants may cause diseases via epigenetic modifications. Epigenetic mechanisms such as DNA methylation participate in the regulation of gene transcription. Surprisingly, epigenetics research is still limited in ecotoxicology. In this study, we investigated whether chronic exposure to contaminants experienced by wild female fish (Anguilla anguilla) throughout their juvenile phase can affect the DNA methylation status of their oocytes during gonad maturation. Thus, fish were sampled in two locations presenting a low or a high contamination level. Then, fish were transferred to the laboratory and artificially matured. Before hormonal treatment, the DNA methylation levels of the genes encoding for the aromatase and the receptor of the follicle stimulating hormone were higher in contaminated fish than in fish from the clean site. For the hormone receptor, this hypermethylation was positively correlated with the contamination level of fish and was associated with a decrease in its transcription level. In addition, whereas gonad growth was associated with an increase in DNA methylation in fish from the clean site, no changes were observed in contaminated fish in response to hormonal treatment. Finally, a higher gonad growth was observed in fish from the reference site in comparison to contaminated fish.

Changes in the role of the thyroid axis during metamorphosis of the Japanese eel, Anguilla japonica.

To clarify the role of thyroid function during metamorphosis from leptocephalus to glass eel in the Japanese eel, we examined the histology of the thyroid gland and measured whole-body concentrations of thyroid hormones, thyroxine (T4) and triiodothyronine (T3), and thyroid stimulating hormone ß-subunit TSH (TSHß) mRNA expression levels in five stages of artificially hatched eels (leptocephalus, early-metamorphosis, late-metamorphosis, glass eel, and elver). During metamorphosis, the inner colloid of thyroid follicles showed positive immunoreactivity for T4, and both T4 and T3 levels were significantly increased, whereas a small peak of TSHß mRNA level was observed at the early-metamorphosis stage. Similarly, TSHß mRNA levels were highest in the glass eel stage, and then decreased markedly in the elver stage. In contrast to TSHß mRNA expression, thyroid hormones (both T4 and T3) increased further from the glass eel to elver stages. These results indicated that thyroid function in the Japanese eel was active both during and after metamorphosis. Therefore, the thyrotropic axis may play important roles not only in metamorphosis but also in subsequent inshore or upstream migrations.

The regulation of aromatase and androgen receptor expression during gonad development in male and female European eel.

This research investigated the regulation of aromatase and androgen receptor gene expression in the brain-pituitary-gonad (BPG) axis of male and female European eels (Anguilla anguilla) during induced sexual maturation. Complete A. anguilla aromatase (aa-cyp19a1) and partial androgen receptor α and ß (aa-ara and aa-arb) sequences were isolated, and qPCR assays were validated and used for quantification of transcript levels for these three genes. Expression levels of the genes varied with sex, tissue and stage of maturation. aa-arb was expressed at higher levels than aa-ara in the pituitary and gonad in both sexes, suggesting aa-arb is the physiologically most important androgen receptor in these tissues. In the female brain, a decrease in aa-ara and an increase in aa-cyp19a1 were observed at the vitellogenic stage. In contrast, a progressive increase in all three genes was observed in the pituitary and ovaries throughout gonadal development, with aa-arb and aa-cyp19a1 reaching significantly higher levels at the vitellogenic stage. In the male pituitary, a decrease in aa-arb and an increase in aa-cyp19a1 were observed at the beginning of spermatogenesis, and thereafter remained low and high, respectively. In the testis, the transcript levels of androgen receptors and aa-cyp19a1 were higher during the early stages of spermatogenesis and decreased thereafter. These sex-dependent differences in the regulation of the expression of aa-ara, aa-arb and cyp19a1 are discussed in relation to the role of androgens and their potential aromatization in the European eel during gonadal maturation.

Effects of dietary supplementation of Lactobacillus pentosus PL11 on the growth performance, immune and antioxidant systems of Japanese eel Anguilla japonica challenged with Edwardsiella tarda.

The aim of this study was to determine the efficacy of dietary administration of Lactobacillus pentosus PL11 on growth performance and the immune and antioxidant systems in Japanese eel Anguilla japonica challenged with Edwardsiella tarda. A total of 75 Japanese eels (24.63±0.83 g) were grouped into 5 treatment diets which were a control diet (C) without E. tarda and 4 treatment diets with E. tarda challenge, including C for E. tarda challenge (NC), C plus L. pentosus PL11 supplemented diet (108 cfu g⁻¹) (T-PL11), C plus L. pentosus KCCM 40997 supplemented diet (108 cfu g⁻¹) (T-Lp) and C plus Weissella hellenica DS-12 supplemented diet (108 cfu g⁻¹) (T-Wh) for 5 weeks (4 week before and 1 week after challenge). The results showed enhanced growth performance in fish fed the diet containing L. pentosus PL11 compared to others. The growth performance parameters including specific growth rate (SGR) and weight gain (WG), feed intake (FI), feed conversion ratio (FCR) and survival were significantly (P<0.05) higher in fish maintained on L. pentosus PL11 supplemented diet compared to C and NC. T-PL11 group also shows a significant increase in the levels of plasma immunoglobulin M, CAT and SOD activities compared to NC. Hematological parameters and mieloperoxidase were significantly better in fish fed the L. pentosus PL11 supplemented diet than in the control. L. pentosus PL11 supplementation recover the reduced expression of SOD, CAT and heat shock protein 70 genes in liver and intestine in pathogen challenged fishes. In conclusion the result of the current study demonstrated L. pentosus PL11 potential as an alternative to antibiotic supplementation to improve the growth and health performance of Japanese eel (A. japonica).

11-ketotestosterone synchronously induces oocyte development and silvering-related changes in the Japanese eel, Anguilla japonica.

To evaluate the effects of sex steroids on silvering in the Japanese eel, Anguilla japonica, the development of oocytes, eye size, digestive tract, and swim bladder were studied in relation to observations of the profiles of plasma levels of sex steroids (estradiol 17ß, E2; testosterone, T; 11-ketotestosterone; 11-KT) during silvering for each sex and by administrating 11-KT to yellow eels. All steroids examined in the study increased in female eels after silvering had begun, whereas in males, only 11-KT increased significantly, and no statistical differences were found in plasma levels of E2 and T between eels in both developmental stages. 11-KT appeared to induce the early stage of oocyte growth, enlargement of the eyes, degeneration of the digestive tract and the development of the swim bladder. This suggested that 11-KT synchronously accelerates early development of the ovaries and the morphological changes, possibly in adaption to oceanic migration, and that 11-KT is one of the most important factors in early stages of development in the Japanese eel, as it appears to be in other anguillid eels.

Testosterone improves the transition of primary oocytes in artificial maturation eels (Anguilla japonica) by altering ovarian PTEN expression.

In mammals, androgens appear to enhance the development of primary ovarian follicles, but PI3K (phosphoinositide 3-kinases) pathway is well recognized as one of the critical pathways in early follicular development. Roles of the PI3K were revealed by deletion of PTEN (phosphatase and tensin homolog on chromosome 10). PTEN is demonstrated to play an important role in the early stage of follicle development. In the Japanese eel, two forms of PTEN have been cloned, but what their functions on the development of early ovarian follicles are still not clear. The natural blockage and inducible of ovarian development was a benefit to address this question in the eel. Testosterone (T) shows to ameliorate the early ovarian development in the eel. The aims of this study were to elucidate the two forms of PTEN by cellular and physiological criteria and to study the effects of T on the ovarian PTEN production in the exogenous pituitary extracts-stimulated eel. Our results suggested that two forms of PTEN are existing in the Japanese eel, and eel ovarian development corresponded to the decrease in ovarian PTEN expression, vice versa. In addition, the supplement of T on eel early ovarian development can be attributed to its PTEN inhibitor role.

Geographic variation in life cycle strategies of a progenetic trematode.

Numerous parasite species have evolved complex life cycles with multiple, subsequent hosts. In trematodes, each transmission event in multi-host life cycles selects for various adaptations, one of which is facultative life cycle abbreviation. This typically occurs through progenesis, i.e., precocious maturity and reproduction via self-fertilization within the second intermediate host. Progenesis eliminates the need for the definitive host and facilitates life cycle completion. Adopting a progenetic cycle may be a conditional strategy in response to environmental cues related to low probability of transmission to the definitive host. Here, the effects of environmental factors on the reproductive strategy of the progenetic trematode Stegodexamene anguillae were investigated using comparisons among populations. In the 3-host life cycle, S. anguillae sexually reproduces within eel definitive hosts, whereas in the progenetic life cycle, S. anguillae reproduces by selfing within the metacercaria cyst in tissues of small fish intermediate hosts. Geographic variation was found in the frequency of progenesis, independent of eel abundance. Progenesis was affected by abundance and length of the second intermediate fish host as well as encystment site within the host. The present study is the first to compare life cycle strategies among parasite populations, providing insight into the often unrecognized plasticity in parasite developmental strategies and transmission.

Androgens and very low density lipoprotein are essential for the growth of previtellogenic oocytes from Japanese eel, Anguilla japonica, in vitro.

To investigate the regulation of lipid uptake into the eel oocyte in more detail, effects of 11-ketotestosterone (11-KT) and lipid transporters (lipoproteins) were determined in vitro. Ovarian explants from previtellogenic Japanese eels (Anguilla japonica) were incubated for 28 days with 11-KT and/or with very low density lipoproteins (Vldl), low density lipoproteins (Ldl), or high density lipoproteins (Hdl) purified from eel plasma. The androgen 11-KT induced notable increases in oocyte diameter, which were accompanied by the appearance of vacuoles rather than lipid. Ldl and Hdl increased oocyte diameters, whereas Vldl did not. However, coincubation of 11-KT and Vldl, but not of Ldl or Hdl, resulted in dramatic increases in oocyte size and lipid droplet surface area. Effects of both 11-KT (oocyte size) and Vldl (lipid droplet surface area) were dose-dependent between 1 and 100 ng/ml and between 0.5 and 5 mg/ml, respectively. Interestingly, abnormal oocyte cytology under conditions of coculture with 11-KT and Vldl could essentially be prevented if Vldl concentrations were high enough (≥ 5 mg Vldl/ml medium). Unlike 11-KT, estradiol-17beta had no effect on oocyte diameter or lipid droplet surface area. We conclude that Vldl is a key transporter of neutral lipids that accumulate into the eel oocyte during oogenesis and that Vldl-dependent lipid uptake is stimulated by the androgen 11-KT.

Molecular characterization of marbled eel (Anguilla marmorata) gonadotropin subunits and their mRNA expression profiles during artificially induced gonadal development.

Three cDNA sequences encoding the gonadotropin subunits, common glycoprotein alpha subunit (GTHalpha), FSHbeta and LHbeta subunits were isolated from marbled eel. The cDNA of GTHalpha encodes 116 amino acids with a signal peptide of 24 amino acids and a mature peptide of 92 amino acids. The FSHbeta subunit consists of 127 amino acids with a 22 amino acid signal peptide and a 105 amino acid mature peptide, while the LHbeta subunit consists of 140 amino acids with a 24 amino acid signal peptide and a 116 amino acid mature peptide. Comparison of the deduced amino acid sequences of marbled eel GTHalpha, FSHbeta, and LHbeta with that of other fishes shows a high degree of conservation in the number of cysteine residues and potential N-linked glycosylation sites. The mRNA of GTHalpha, FSHbeta and LHbeta were not only detected in pituitary, but also in ovary and testes by RT-PCR. Quantitative realtime PCR analysis revealed that the GTHalpha and LHbeta transcriptional levels in pituitaries of female and male eels gradually increased during the artificially inducing gonadal development, and peaked at late vitellogenic stage and spermiation stage, respectively. FSHbeta mRNA in the pituitaries of female eels maintained a high level at previtellogenic stage, early vitellogenic stage as well as mid-vitellogenic stage but declined sharply at late vitellogenic stage and migratory nucleus stage. In male eels, the mRNA levels of FSHbeta in the pituitaries were higher at early spermatogenesis stage than at both late spermatogenesis stage and spermiation stage. These results suggested that FSH would be in control of initiation and maintenance of gonadal growth and gametogenesis, whereas LH would be involved in the final gonadal maturation and spermiation/ovulation in the tropic eel Anguilla marmorata.

Yellow eel (Anguilla anguilla) development in NW Portuguese estuaries with different contamination levels.

The aims of the present study were to compare the health status of yellow eels (Anguilla anguilla) developing in three estuaries of the NW Portuguese coast with different levels of pollution and their physiological responses to combined effects of environmental variation and pollution. For this, a field study was performed using a multi-parameter approach, including eels condition indexes and biomarkers, water quality variables and other environmental factors. Sixteen biological parameters were assessed, namely: hepatosomatic index (LSI), Fulton's condition index (K), lipid peroxidation (LPO), total glutathione (TG), reduced glutathione (GSH), oxidised glutathione (GSSG), GSH/GSSG, and the activity of the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH), sodium-potassium ATPase (Na(+)/K(+)-ATPase), ethoxyresorufin O-deethylase (EROD), glutathione S-transferases (GST), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR). Ten environmental factors were also measured in water: temperature, salinity, pH, phosphates, nitrates, nitrites, ammonium, silica, phenol and hardness. Globally, the biomarkers indicate exposure and toxic effects of pollutants on eels living in contaminated estuaries. The relationships between biological and environmental variables were assessed through redundancy analysis. K and LSI indexes, AChE and Na(+)/K(+)-ATPase, total glutathione levels and the antioxidant enzymes CAT, GR, and SOD where the factors most discriminating reference (Minho River estuary) from contaminated estuaries (Lima and Douro Rivers estuaries). Moreover, the most striking outcomes of pollutants exposure on biological responses were observed during winter, probably due to a joint effect of cold weather and pollution stress. Altogether, the results indicate that the development of eels in the polluted estuaries of Lima and Douro rivers is interfering with physiological functions determinant for their survival and performance. This may increase the mortality rates during the continental life-phase of the species and decrease the percentage of animals able to successfully complete their oceanic migration and, thus, reduce the contribution of each generation to the next one.

Changes in gene expression in European eels (Anguilla anguilla) induced by infection with swim bladder nematodes (Anguillicola crassus).

The European eel, Anguilla anguilla, is considered an endangered species that is under pressure for many reasons. Among others, the introduced parasite Anguillicola crassus is thought to play an important role in the decline of eel populations. These nematodes have been shown to negatively affect many fitness-related traits in eels, e.g., growth, osmoregulation, and stress tolerance. Nevertheless, there has been little work on the way in which the host-parasite interaction influences the molecular regulation of these key physiological processes. We experimentally analyzed the effect of this nematode on the expression of genes involved in the physiology of European eels during their continental life. Included are genes that are implicated in the eel's somatic growth (insulin-like growth factor 1 and thyroid hormone receptor beta), osmoregulation (Na+/K+-ATPase beta1 and aquaporin 3), and hematopoiesis (hemoglobin alpha-chain). Our results showed the absence of an effect on genes involved in fish growth; the parasite may, however, have an effect on osmoregulation and hematopoiesis. We also noted a differential impact of male and female parasites on the expression of some genes, perhaps owing to the sexual dimorphism in body size of the parasite.

Male silver eels mature by swimming.

BACKGROUND: If European silver eels are prevented from reproductive migration, they remain in a prepubertal stage by dopaminergic inhibition of pituitary activity. Because this inhibition is likely a requirement for an extended female growth stage, we tested if it is sex-specific by subjecting both sexes to stimulation by GnRHa (Gonadotropin-Releasing Hormone agonist) - injection or 3-months swimming in seawater. RESULTS: In contrast to females, males showed a two- to three-fold higher LHbeta (luteinising hormone beta subunit) - expression, a three- to five-fold higher GSI (Gonadosomatic index) and induced spermatogenesis when compared with the untreated control group. CONCLUSION: Dopaminergic inhibition is thus not effective in males and swimming results in natural maturation, probably via GnRH-release.

Molecular cloning and gene expression of Spo11 during spermatogenesis in the Japanese eel, Anguilla japonica.

Spo11 is a protein involved specifically in the meiotic recombination in several species, however, it is little characterized in lower vertebrates. We identified a cDNA encoding Spo11 from the testis of Japanese eel, Anguilla japonica. The deduced amino acid sequence of eel Spo11 was more than 60% identical with human and mouse Spo11s. In order to examine changes in the expression and localization of Spo11 during spermatogenesis induced by the injection of human chorionic gonadotropin (hCG) and to compare with those of Dmc1, we generated specific antibodies against the eel Spo11 and Dmc1. In general, it is believed that Dmc1 is a meiosis-specific protein, and the localization of Dmc1 in spermatocytes was confirmed also in Japanese eel. Spo11 transcripts were slightly detected in the testis after 1 day post-hCG injection by Northern blot analysis. Western blot analysis also indicated that Spo11 production began at day 1 after hCG injection. However, immunohistochemical observations showed that Spo11 was localized only in spermatocytes. In contrast, Dmc1 transcripts and the protein production were first detected at day 6 after hCG injection and increased along with the increment of spermatocytes. These results suggested that Spo11 was expressed in spermatogonia proliferated toward meiosis at quite low level that could not induce meiotic recombination, thereafter Spo11 expression increased and Dmc1 expression was initiated in early meiotic prophase. Hence, the antibodies against eel Spo11 and Dmc1 generated in the present study can be use to detect germ cells in early meiotic prophase immunohistochemically. Importantly, it is suggested that germ cells, which are in quite earlier stage during meiotic prophase, can be detected by Spo11.

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-17beta (E(2)) 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 E(2) in females increased significantly during silvering (P<0.05), while E(2) 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 E(2) and T concentrations increased with ovarian development of wild Japanese eels during silvering, while serum E(2) 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.