Prostaglandin production by human epidermal cells in vitro: a model for studying pharmacologic inhibition of prostaglandin synthesis

Blood prostaglandin A levels were measured in 10 healthy subjects under different conditions of collection and storage. Plasma levels ranged from 1.20 to 1.81 ng/ml (M + or - SE=1.50 + or - 0.10) in 5 females, to 1.23 to 1.68 ng/ml (1.45 + or - 0.09) in 5 males, when centrifuged and frozen immediately after collection. Storage at 4 degrees Centigrade (C) for varying times up to 24 hours and at 22 degrees C (room temperature) up to 4 hours did not affect mean plasma concentrations significantly, but increased the range obtained to 1.00 to 2.47 ng/ml for both male and female groups. Serum concentrations differed in males and females and were lower than corresponding mean plasma values for males and higher for females. Mean serum concentrations were 1.77 + or - 0.08 ng/ml in females and 1.18 + or - 0.05 ng/ml in males and did not change significantly up to 24 hours of storage at 4 decrees C. These results suggest that prostaglandin A assayed in both plasma and serum under the conditions described is stable and should allow for greater flexibility in sampling under different experimental conditions.     

Development of an enzyme-linked immunosorbent assay for vitellogenin of Morelet's crocodile (Crocodylus moreletii)

The purpose of this study was to develop an immunoassay for vitellogenin in Morelet's crocodile (Crocodylus moreletii). Blood was collected from wild-caught crocodiles in Belize. Plasma samples from adult females taken during the breeding season were used for vitellogenin purification and samples from adult males were used for comparison. No differences were detected between males and females for plasma total protein concentration, as measured by Coomassie assay. However, denaturing polyacrylamide gel electrophoresis (SDS-PAGE) revealed that female plasma contained a 210-kDa protein, presumably the vitellogenin monomer, that was absent in adult male plasma. The identity of the putative vitellogenin was confirmed by its cross-reactivity in Western blots with a vitellogenin antiserum that was generated against a conserved vitellogenin peptide sequence. Crocodile vitellogenin was purified by two successive rounds of DEAE chromatography. The purified protein had an apparent molecular mass of 450 kDa, as determined by gel filtration chromatography, and 210 kDa on SDS-PAGE. An indirect enzyme-linked immunosorbent assay (ELISA) was then developed for C. moreletii vitellogenin. The detection limit of the assay was 20.0 ng/mL. The intra- and inter-assay coefficients of variation were 5.3% and 9.8%, respectively. The recovery of vitellogenin diluted into male plasma was 94.7%. The ELISA assay revealed that vitellogenin levels of adult female plasma during the breeding season ranged from 1.8 to 3.1 mg/mL with a mean of 2.5+/-0.25 mg/mL. No vitellogenin was detected in adult male plasma. Induction of vitellogenin in Morelet's crocodile may be a useful model system for field studies of crocodile reproduction and for investigations of endocrine disruption in this species.     

Blood prostaglandin A (PGA) levels in normal human subjects

Blood prostaglandin A levels were measured in ten healthy subjects under different conditions of collection and storage.Plasma levels ranged from 1.20 to 1.81 ng/ml (M ± SE = 1.50 ± 0.10) in 5 females to 1.23 to 1.68 ng/ml (1.45 ± 0.09) in 5 males, when centrifuged and frozen immediately after collection. Storate at 4°C for varying times up to 24 hours and at 22°C (room temperature) up to 4 hours did not affect mean plasma concentrations significantly, but increased the range obtained to 1.00 to 2.47 ng/ml for both male and female groups.Serum concentrations differed in males and females and were lower than corresponding mean plasma values for males and higher for females. Mean serum concentrations were 1.77 ± 0.08 ng/ml in females and 1.18 ± 0.05 ng/ml in males and did not change significantly up to 24 hours of storage at 4°C.These results suggest that prostaglandin A assayed in both plasma and serum under the conditions described is stable and should allow for greater flexibility in sampling under different experimental conditions.     

Macrofilaricidal and microfilaricidal effects of Neurolaena lobata, a Guatemalan medicinal plant, on Brugia pahangi

Twelve extracts of 11 Guatemalan medicinal plants were initially screened in vitro for potential macrofilaricidal activity against Brugia pahangi, a lymphatic dwelling filarial worm, using concentrations from 125 to 1000 microg ml(-1) of each extract that could be dissolved in the culture medium. Of 12 extracts used, the ethanol extract of leaves of Neurolaena lobata showed the strongest activity against the motility of adult worms. Subsequently, the extract of N. lobata was extensively examined in vitro for macro- and micro-filaricidal effects using a series of concentrations of 500, 250, 100, 50 and 10 microg ml(-1). The effects were assessed by worm motility, microfilarial release by female worms and a MTT assay. The effect on the motility of adult worms was observed in a concentration- and time-dependent manner. The time required to stop motility of both sexes of adult worms was 6 h at 500 microg ml(-1), 24 h at 250 microg ml(-1), and 3 days for females and 4 days for males at 100 microg ml(-1). The movement of females ceased at 4 days at a concentration of 50 microg ml(-1) whereas the motility of males was only reduced. The loss of worm's viability was confirmed by the MTT assay and was similar to the motility results. These concentrations, including 10 microg ml(-1), prevented microfilarial release by females in a concentration- and time-dependent manner. Concentrations higher than 100 microg ml(-1) even induced mortality of the microfilariae. The present study suggested that the ethanol extract of Neurolaena lobata has potential macro- and micro-filaricidal activities.     

Monoclonal antibody-based immunoassay of vitellogenin in the blood of male channel catfish (Ictalurus punctatus).

1. A monoclonal antibody to vitellogenin of channel catfish (Ictalurus punctatus) was made, and its specificity was demonstrated using Western blots of serum from female fish, estradiol-treated male fish, untreated male fish, vitellogenin purified by three different methods and egg extracts. 2. An enzyme-linked immunosorbent assay (ELISA), using this monoclonal antibody, detected vitellogenin in the plasma of 59 out of 60 untreated 17-24-month-old male channel catfish with a mean concentration of 338 micrograms/ml and a maximum concentration of 4240 micrograms/ml. 3. Vitellogenin levels in male channel catfish were unrelated to testicular stage, gonadosomatic index and month.     

Serum thyroxine and triiodothyronine radioimmunoassay values in the normal ferret

The European ferret, Mustela putorius furo, has become increasingly popular as an animal model in biomedical research. However, certain important normal clinical data have not been established for the ferret. In this study, serum thyroxine (T4) and 3,3',5-triiodothyronine (T3) values were obtained from ferrets by the use of commercial radioimmunoassays. Sera from 44 animals, 31 males (27 intact and 4 castrated) and 13 females (10 intact and 3 spayed) were assayed. Serum T4 values ranged from 1.01-8.29 micrograms/dl for males (mean = 3.24 +/- 1.65 micrograms/dl), and 0.71-3.43 micrograms/dl for females (mean = 1.87 +/- 0.79 micrograms/dl). Serum T4 values of adult female ferrets, juvenile ferrets (less than 1 year old) of either sex, and castrated males were similar to the normal T4 values of the cat, 1.20-3.80 micrograms/dl. Intact adult male ferrets had higher serum T4 values which were more comparable to those of the normal dog 1.52-3.60 micrograms/dl. Serum T3 values ranged from 0.45-0.78 ng/ml for males (mean = 0.58 +/- 0.09 ng/ml), and 0.29-0.73 ng/ml for females (mean = 0.53 +/- 0.13 ng/ml). These values are comparable to those of dogs and cats which are 0.50-1.50 ng/ml.     

Bioactive follicle-stimulating hormone levels in serum and urine of male and female rats from birth to prepubertal period

Using a sensitive in vitro granulosa cell aromatase bioassay (GAB), we determined serum and urinary levels of bioactive follicle-stimulating hormone (bio-FSH) in male and female rats from birth to Day 40 of age. In addition, serum immunoreactive FSH (immuno-FSH) was measured by radioimmunoassay to determine the bio- to immuno-(B/I) ratio of FSH. During the neonatal period (Days 1-7 of age), both sexes had detectable serum bio-FSH levels. In the infantile period (Days 7-21), serum bio-FSH levels initially decreased at Day 10 for both sexes, and then rose steadily, reaching maximum concentrations at Day 14 (males: 68.7 ng/ml; females: 114.6 ng/ml). Subsequently, FSH levels in the females decreased from Day 16 throughout the juvenile (Days 21-35) and prepubertal (Days 35-40) periods. In contrast, FSH levels in the males fluctuated during these periods. In the males, immuno-FSH reflected the bioactive profiles, with a B/I ratio of 2.2 +/- 0.2. In the females, the B/I ratio was approximately 2.5 during the neonatal and infantile periods but declined to approximately 1.0 during the juvenile and prepubertal periods, consistent with earlier observations of heterogeneous forms of pituitary FSH in immature female rats. Morning urine samples were also collected daily and bio-FSH concentrations were determined. In both sexes, urinary bio-FSH profiles were highly correlated (r = 0.93) with serum FSH throughout development. However, the urine concentrations were about 50-fold higher than serum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of vitellogenin from smooth flounder (Pleuronectes putnami) and measurement in plasma by homologous ELISA

Male smooth flounder (Pleuronectes putnami) were induced to produce vitellogenin (VTG) by injection of 17beta-estradiol (E2). Anion exchange chromatography of precipitated plasma from E2-injected resulted in a single peak consisting of VTG. Smooth flounder VTG has an approximate molecular mass of approximately 520 kDa, determined by gel filtration with molecular weight standards. Purified VTG was used to develop a homologous enzyme-linked immunosorbent assay (ELISA). The flounder VTG ELISA is an indirect antigen competition assay with a detection limit of 15 ng.ml(-1) and a useful range of 30-950 ng.ml(-1) of diluted sample. Intra- and inter-assay precision (as %CV, n=7) ranged from 1.3% to 6.0% and 5.1%, respectively. The ELISA was evaluated using plasma samples collected from a smooth flounder population captured in the Saint Lawrence Estuary. The ELISA is sensitive enough to differentiate males and non-vitellogenic females from vitellogenic individuals during early vitellogenesis.     

Induction of vitellogenesis by estradiol-17beta and development of enzyme-linked immunosorbant assays to quantify plasma vitellogenin levels in green turtles (Chelonia mydas)

Treatment of juvenile green turtles (Chelonia mydas) with estradiol-17beta resulted in the induction of a 200 kDa plasma protein, consistent with vitellogenin (Vtg). The N-terminal 15 amino acids of the anion exchange purified protein shared sequence homologies with vitellogenins of several vertebrate species. Rabbit antiserum raised against purified Vtg recognized the plasma protein as well as several yolk proteins. Monoclonal antibody (Mab) HL1248, produced by inoculating mice with turtle yolk granules, showed specificity for plasma Vtg as well as a set of yolk proteins 120, 82, 43 and 32 kDa in size. The N-terminal 22 amino acids of the 43 kDa yolk protein was similar to the lipovitellin I subunit of Vtg of several vertebrate species. The peptide mass map of the 82 kDa yolk protein shared enough ions with that of purified plasma Vtg to support the conclusion that this protein was derived from plasma Vtg. Taken together, these results validate the specificity of Mab HL1248 for Vtg. Using purified Vtg concentration standards, competition and antigen capture enzyme-linked immunosorbant assays (ELISAs) were shown to quantitatively detect Vtg in green turtle plasma. Pre-induced plasma of juvenile turtles had Vtg levels of 2-4 micrograms/ml whereas post-estradiol exposure samples had 38-40 mg/ml. The plasma Vtg concentration of a nesting female turtle was 4.6 mg/ml, approximately 20-fold higher than that of a non-nesting adult female. The antigen capture ELISA will be useful in population studies of this endangered species, to detect vitellogenesis in females that will nest in a given year and to detect inappropriate Vtg levels in turtles exposed to xenoestrogens.     

Determination of UDP-N-acetylglucosamine:β-D-mannoside-1,4-N-acetylglucosaminyltransferase-III in patients sera with chronic hepatitis and liver cirrhosis using a monoclonal antibody

The glycoprotein UDP-N-acetylglucosamine: beta-D-mannoside-1,4-N-acetylglucosaminyltransferase-III (GnT-III) catalyzes the addition of N-acetylglucosamine via a beta-1, 4-linkage to the beta-linked mannose of the trimannosyl core of N-linked glycans. It has been reported that the expression of GnT-III increases in many oncogenically transformed cells and human hepatocellular carcinoma (HCC) tissues, and GnT-III enzyme activity in serum can be used for the detection and monitoring of primary hepatomas and hepatocellular carcinomas. A solid-phase enzyme-linked immunosorbent sandwich assay in which a polyclonal antibody (PAb) to aglycosylrecombinant GnT-III (AGR-GnT-III) and a monoclonal antibody (mAb) are employed as a capture protein and probe protein, respectively, is described. The sensitivity of the PAb-mAb sandwich assay, as determined by the dose-response effect for AGR-GnT-III, was 10 ng/ml. This assay was specific for GnT-III and did not detect beta-1, 6-N-acetylglucosaminyltrasferase-V (GnT-V). AGR-GnT-III concentrations in 377 serum specimens were determined by the PAb-mAb sandwich assay and the results were analyzed based on the disease category, using 1.99 microg/mL (AGR-GnT-III) as a cut-off value. The AGR-GnT-III level of 61 normal serum samples was 0.57 +/- 0.71 microg/ml (mean +/- SD). The results revealed an elevation in serum AGR-GnT-III levels in 60 of 86 patients (3.03 +/- 2.04 microg/ml) with liver cirrhosis (LC) and 86 of 91 patients (2.73 +/- 0.59 microg/ml) with chronic hepatitis (CH). By contrast, 3 of 61 normal subjects, 9 of 34 patients (1.02 +/- 1.03 microg/ml) with acute hepatitis and 8 of 38 patients (1.79 +/- 0.56 microg/ml) with a variety of non-hepatic diseases exhibited a slight increase above the cut-off value. These results indicate that serum AGR-GnT-III levels are elevated predominantly in LC or CH cases. Serum AGR-GnT-III concentration, as measured by the developed PAb-mAb sandwich assay, may be a useful differential marker as a diagnostic aid for CH and/or LC and warrants further investigations with expanded serum panels.     

Serum molybdenum concentration in healthy Japanese adults determined by inductively coupled plasma-mass spectrometry

The serum molybdenum (Mo) concentrations in 70 Japanese adults (35 males and 35 females) not receiving any medical care or treatment were determined by inductively coupled plasma-mass spectrometry. Serum Mo concentration in the subjects ranged from <0.1 to 9.11 ng/mL. More than half (55.7%) of the subjects showed values of less than 1 ng/mL and only 6 (8.6%) subjects showed more than 2 ng/mL. The mean+/-SD, geometrical mean (GM), range of GM+/-geometrical SD (GSD) and median value were 1.21+/-1.34, 0.81, 0.30 to 2.16, and 0.90 ng/mL, respectively. Among age, body mass index and several serum biochemical values, activities of aspartate aminotransferase and alanine aminotransferase showed significant associations with serum Mo; 15 subjects suspected of having liver dysfunction showed significantly higher serum Mo than others. We propose a range of 0.10-4.73 ng/mL, estimated as a range of GM+/-2GSD of serum Mo in the remaining 55 subjects without liver dysfunction, as a reference range of serum Mo in Japanese healthy adults.     

First Assessment of the Sex Ratio for an East Pacific Green Sea Turtle Foraging Aggregation: Validation and Application of a Testosterone ELISA

Determining sex ratios of endangered populations is important for wildlife management, particularly species subject to sex-specific threats or that exhibit temperature-dependent sex determination. Sea turtle sex is determined by incubation temperature and individuals lack external sex-based traits until sexual maturity. Previous research utilized serum/plasma testosterone radioimmunoassays (RIA) to determine sex in immature/juvenile sea turtles. However, there has been a growing application of enzyme-linked immunosorbent assay (ELISA) for wildlife endocrinology studies, but no study on sea turtles has compared the results of ELISA and RIA. This study provides the first sex ratio for a threatened East Pacific green sea turtle (Chelonia mydas) foraging aggregation, a critical step for future management of this species. Here, we validate a testosterone ELISA and compare results between RIA and ELISA of duplicate samples. The ELISA demonstrated excellent correspondence with the RIA for providing testosterone concentrations for sex determination. Neither assay proved reliable for predicting the sex of reproductively active females with increased testosterone production. We then applied ELISA to examine the sex ratio of 69 green turtles foraging in San Diego Bay, California. Of 45 immature turtles sampled, sex could not be determined for three turtles because testosterone concentrations fell between the ranges for either sex (females: 4.1–113.1 pg/mL, males: 198.4–2,613.0 pg/mL) and these turtles were not subsequently recaptured to enable sex determination; using a Bayesian model to predict probabilities of turtle sex we predicted all three ‘unknowns’ were female (> 0.86). Additionally, the model assigned all turtles with their correct sex (if determined at recapture) with 100% accuracy. Results indicated a female bias (2.83F:1M) among all turtles in the aggregation; when focusing only on putative immature turtles the sex ratio was 3.5F:1M. With appropriate validation, ELISA sexing could be applied to other sea turtle species, and serve as a crucial conservation tool.     

Vitellogenin Titres in Normal and Accelerated Maturation of Gregarious-Phase Schistocerca gregaria

Isolation of vitellogenin of the Schistocerca gregaria (Forskal) in its gregarious phase was achieved by a combination of gel permeation and anion exchange chromatography. Staining for carbohydrate and lipid moieties showed that the vitellogenin is a glycolipoprotein. The vitellogenin of S. gregaria has a native molecular weight of about 700 kDa. On SDS-PAGE, the protein showed nine apoproteins of about 124, 120, 105, 60, 59, 58, 57, 53 and 34 kD. Determination of the levels of vitellogenin by ELISA in the haemolymph of maturing females showed that those exposed to mature males from 1 to 2 days after ecdysis had increased levels of vitellogenin from day 10 (81.1 ± 4.5). In contrast, females exposed to immature males or kept alone showed an increase (107.3 ± 0.9 and 70.2 ± 2.7) not until day 16 or later, respectively. These results are consistent with the accelerating effect of pheromonal emissions from mature males on the maturation of female S. gregaria.     

Effect of estrogenic activity, and phytoestrogen and organochlorine pesticide contents in an experimental fish diet on reproduction and hepatic vitellogenin production in medaka (Oryzias latipes)

Endocrine-disrupting chemicals (EDCs) are giving rise to serious concerns for humans and wildlife. Phytoestrogens, such as daidzein and genistein in plants, and organochlorine pesticides are suspected EDCs, because their chemical structure is similar to that of natural or synthetic estrogens and they have estrogenic activity in vitro and in vivo. We assessed estrogenic activity and dietary phytoestrogen and organochlorine pesticide contents of various fish diets made in the United Kingdom, and compared them with those features of diets made in Japan that were tested in a previous study. Genistein and daidzein were detected in all of the diets. Using an in vitro bioassay, many of these diets had higher activation of estrogen beta-receptors than estrogen alpha-receptors. Organochlorine pesticides such as hexachlorobenzene, beta-benzene hexachloride (BHC), and gamma-BHC were detected in all fish diets. On the basis of these data, we investigated the effect of differing dietary phytoestrogen content in Japanese fish diets on hepatic vitellogenin production and reproduction (fecundity and fertility) in medaka (Oryzias latipes). Assessment of the effects of a 28-day feeding period on reproduction of paired medaka did not indicate significant differences in the number of eggs produced and fertility among all feeding groups. However, hepatic vitellogenin values were significantly higher for male medaka fed diet C (genistein, 58.5 +/- 0.6 microg/g; daidzein, 37.3 +/- 0.2 microg/g) for 28 days compared with those fed diet A (genistein, < 0.8 microg/g; daidzein, < 0.8 microg/g) or diet B (genistein, 1.4 +/- 0.1 microg/g; daidzein, 2.0 +/- 0.1 microg/g). Our findings indicate that fish diets containing high amounts of phytoestrogens, such as diet C, have the potential to induce hepatic vitellogenin production in male medaka, even if reproductive parameters are unaffected. Therefore, some diets, by affecting vitellogenin production in males, may alter estrogenic activity of in vivo tests designed to determine activity of test compounds added to the diet.     

Synthesis and antifungal activity of substituted-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles

Series of substituted-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles derivatives have been synthesized and examined for their activity against pathogenic strains of Aspergillus fumigatus (ITCC 4517), Aspergillus flavus (ITCC 5192) Aspergillus niger (ITCC 5405) and Candida albicans (ITCC No 4718). All synthesized compounds showed mild to moderate activity, except for 2-substituted-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles 6a-d. The most active 1-(4-chlorophenyl)-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indole 4c exhibited a MIC value of 5.85 microg/disc against A. fumigatus and 11.71 microg/disc against A. flavus and A. niger in disc diffusion assay. Anti-Aspergillus activity of active compound 4c by microbroth dilution assay was found to be 15.62 microg/ml in case of A. fumigatus and 31.25 microg/ml with A. flavus and A. niger. The MIC90 value of the most active compound by percent germination inhibition assay was found to be 15.62 microg/ml against A. fumigatus. The MIC90 values of substituted-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles against C. albicans ranged from 15.62 to 250 microg/ml. The in vitro toxicity of the most active 1-(4-chlorophenyl)-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indole 4c was evaluated using haemolytic assay, in which the compound was found to be non-toxic to human erythrocytes up to a concentration of 312.50 microg/ml. The standard drug amphotericin B exhibited 100% lysis at a concentration of 37.5 microg/ml.     

Reproductive effects of prenatal exposure to nonylphenol on zebrafish (Danio rerio)

Mature female and male zebrafish were separated and exposed to nonylphenol (NP) at 0.1, 1, 10, 50, 100 and 500 microg/L, respectively, for 3 weeks. Gonadosomatic index (GSI) in both sexes and vitellogenin (VTG) induction in males was measured as the bioindicators for the impairment to the parents. The results indicated that 50 microg/L of NP was the non-observed effect concentration (NOEC) for GSI and VTG induction. Afterwards, the 50 microg/L NP exposed females and males, and the control females and males were cross-wise pair-bred in the control water for one week to examine the reproductive effects. The embryonic cathepsin D (CAT D) activity, eggshell thickness, fecundity, hatching rate and malformation (vertebral column flexure) rate of offspring were determined in the four pair-bred groups. While endpoints remained unchanged in the groups with exposed males, prenatal exposure of females to 50 microg/L of NP resulted in the impairment of reproduction in groups with exposed females including inhibition of CAT D activity (P < 0.05), decrease of eggshell thickness (by 23.6%) and elevation of malformation rate (P < 0.001). These results suggested NP could induce reproductive damage to zebrafish at NOEC for parents. The results also imply that alterations of CAT D activity and eggshell thickness may be more sensitive biomarkers to indicate the reproductive effects caused by endocrine disrupting chemicals.     

维生素E对中华鳖幼鳖生长、肝脏维生素E以及血清皮质醇含量的影响

为探讨维生素E(VE)对中华鳖(Pelodiscus sinensis)幼鳖的生长、肝脏VE和血清皮质醇的影响，通过特定生长率、高压液相色谱法和放免法，我们测定了中华鳖幼鳖的生长、肝脏VE和血清皮质醇含量。发现VE添加量为1000和5000mg／kg的两组，能明显降低中华鳖幼鳖的生长。维生素E添加量为500、1000和5000mg／kg的三组，肝脏维生素E含量明显高于对照组，VE添加量在0—1000mg／kg的范围时，肝脏VE的含量随着饲料中VE含量的增加呈指数式增加，并且在VE添加量为5000kg／kg的一组基本达到饱和。维生素E添加量为0和50mg／kg的2组，其血清皮质醇的平均值明显高于维生素E添加量为250、500、1000和5000mg／kg的4组的平均值。上述结果表明：高剂量的VE降低了中华鳖幼鳖的生长和血清皮质醇的含量；在一定剂量范围内，肝脏VE随着饲料中VE含量的增加而升高。     

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.     

Toxicity of the aromatase inhibitor letrozole to Japanese medaka (Oryzias latipes) eggs, larvae and breeding adults

Letrozole is a synthetic aromatase inhibitor and interferes in the committed step in the synthesis of endogenous estrogens from androgens. To evaluate potential effects on the early life stages of Japanese medaka, larvae and fertilized eggs were exposed to letrozole for 96 h and 14 days, respectively. No larvae died and no adverse effects were found on embryonic development at concentrations up to 3125 microg/L. Reproductive effects were assessed by exposing adults to 1, 5, 25, 125 and 625 microg/L letrozole for 21 days. A dose-dependent decrease in fecundity (>25 microg/L) and fertility (>5 microg/L) accompanied by histological changes suggested the inhibition of oocyte growth and possibly maturation. At 625 microg/L, the fish ceased spawning during the last week of exposure. Letrozole (>5 microg/L) reduced plasma vitellogenin levels in females in a dose-dependent manner. Transgenerational effects were evaluated by removing freshly-laid F1 eggs from letrozole-contaminated water and raising them to 15 days post-hatching in control water. Hatchability and time to hatching were detrimentally affected (>5 microg/L), but no morphological deformities were observed. Furthermore, a dose-dependent increase in the proportion of genotypic F1 males was found (>5 microg/L).