Kiyoshi Soyano

Effect of some vertebrate and invertebrate hormones on the population growth, mictic female production, and body size of the marine rotifer Brachionus plicatilis Müller

Eight vertebrate and invertebrate hormones were screened for theireffect on population growth, mictic female production, and body size of themarine rotifer Brachionus plicatilis. Growth hormone (GH) or human chorionicgonadotropin (HCG) at 0.0025–25 I.U. ml−1 andestradiol-17β (E2), triiodothyronine (T_3),20-hydroxyecdysone (20-HE), 5-hydroxytryptamine (5-HT), gamma-aminobutyricacid (GABA) or juvenile hormone (JH) at 0.05–50 mg l−1were added to 5-ml of Nannochloropsis oculata suspension (7×106 cells ml−1). From an initial densityof 1 individual ml−1, rotifers were cultured with hormones for48 hours in 22 ppt seawater at 25 °C, in darkness.Rotifers were counted and classified into female types and transferred to anew algal food suspension without hormone every other day until day 8 whenbody size was measured. Population growth was significantly higher intreatments exposed to GABA (50 mg l−1), GH (0.0025 and 0.025I.U. ml−1), HCG (0.25 and 2.5 I.U. ml−1), and5-HT (5 mg l−1). E2 caused a decrease inpopulation growth, whereas JH, 20HE, and T3 had no effect.Mictic female production was significantly higher at 0.05 and 0.5 mgl−1 JH and 0.05 and 5 mg l−1 5HT. GH (0.0025 and0.025 I.U. ml−1), E2 (50 mg l−1),GABA (0.5, 5 and 50 mg l−1), and 20-HE (0.05 mgl−1) treatments had significantly higher mictic femaleproduction only on day 8, 6, 4, and 6, respectively. T3 andhCG had no effect on mictic female production. Lorica length increased by9.6% and 4.4% in rotifers treated with JH (0.05 mgl−1) and GABA (5 mg l−1), respectively. Correspondingly, lorica width increased by 8.9% and 2.6% inthese treatments. In comparison, 20-HE-, T3-, and HCG-treatedrotifers were smaller (3.9–8.2%) and GH, 5-HT andE2 had no effect on rotifer body size.

Induction of Female-to–male Sex Change in the Honeycomb Grouper (Epinephelus merra) by 11-ketotestosterone Treatments

Abstract The honeycomb grouper, Epinephelus merra, is a protogynous hermaphrodite fish. Sex steroid hormones play key roles in sex change of this species. A significant drop in endogenous estradiol- 17β (E2) levels alone triggers female-to–male sex change, and the subsequent elevation of 11-ketotestosterone (11KT) levels correlates with the progression of spermatogenesis. To elucidate the role of an androgen in sex change, we attempted to induce female-to–male sex change by exogenous 11KT treatments. The 75-day 11KT treatment caused 100% masculinization of pre-spawning females. Ovaries of the control (vehicle-treated) fish had oocytes at various stages of oogenesis, while the gonads of the 11KT-treated fish had transformed into testes; these contained spermatogenic germ cells at various stages, including an accumulation of spermatozoa in the sperm duct. In the sex-changed fish, plasma levels of E2 were significantly low, while both testosterone (T) and 11KT were significantly increased. Our results suggest that 11KT plays an important role in sex change in the honeycomb grouper. Whether the mechanism of 11KT-induced female-to–male sex change acts through direct stimulation of spermatogenesis in the ovary or via the inhibition of estrogen synthesis remains to be clarified.

Survey of contamination of estrogenic chemicals in Japanese and Korean coastal waters using the wild grey mullet (Mugil cephalus)

We monitored the contamination by environmental estrogens (EEs) of coastal areas in Korea and Japan using the wild grey mullet. The grey mullet were collected from Ansan, Jeju, Yeosu, Tongyeong, and Busan in Korea and Nagasaki, Omuta, and Fukuoka in Japan. Contamination by EEs was determined by measuring vitellogenin (VTG) levels in serum and identifying gonadal abnormalities histologically (i.e., testis-ova). In four sites in Korea (Ansan, Yeosu, Tongyeong, and Busan) and two sites in Japan (Nagasaki and Fukuoka), serum VTG in immature and male grey mullet was detected at levels greater than 1.0 microg/ml, which is considered to be an abnormal level. Although, testis-ova were observed in some individuals collected in Ansan, Tongyeong, and Busan in Korea and Omuta in Japan, there was no correlation between individuals with testis-ova and individuals with abnormal levels of VTG. Furthermore, in Japan, serum VTG levels of fish collected from Nagasaki and Fukuoka were also greater than 1.0 microg/ml. Although individuals with testis-ova were found in Omuta, these fish expressed normal levels of serum VTG. Our results suggest that the grey mullets living in these coastal areas are influenced by EEs in the environment. Furthermore, it appears that the production of VTG and the occurrence of testis-ova are caused by different mechanisms.

Immunohistochemical evidence identifying the site of androgen production in the ovary of the protogynous grouper Epinephelus merra.

Androgen plays an important role in the developing ovaries of female fish. However, little is known regarding either the sites of production of androgen or its functional roles. In the present study, we investigated immunohistochemically the localization of cholesterol-side-chain-cleavage (P450scc) and cytochrome P45011β-hydroxylase (P45011β) with antibodies P450scc and P45011β in the ovary of the female honeycomb grouper Epinephelus merra during its reproductive cycle. Clusters of strongly immunopositive cells, with 100–1000 cells in each cluster, against both P450scc and P45011β, were observed throughout the annual reproductive cycle in tissue near blood vessels in the tunica ovary surrounding the outer periphery of the ovary. The ultrastructural characteristics of these cells showed that they were steroid-producing cells. In contrast, immunopositive cells against P450scc but not against P45011β were localized in the theca layer surrounding the outer periphery of oocytes. These results suggest that two distinct steroid biosynthesis sites exist in the ovary and that cells at the two sites differ functionally. The only cells that biosynthesize 11-ketotestosterone are found in clusters in the vicinity of blood vessels; they possibly play a physiological role in oocyte growth and gonadal restructuring during the sex change of individuals of this species.

Gonadal morphology in the self-fertilizing mangrove killifish, Kryptolebias marmoratus

We conducted anatomical and histological observations of the gonads in the self-fertilizing mangrove killifish, Kryptolebias marmoratus to investigate the self-fertilizing mechanism of this species. The gonad has a bilobed structure. The elongated gonadal lumen (GL) along the dorsal surface of the gonad connects to the common genital sinus. The elongate testicular region is closely attached to the GL. Among the ovulated eggs in the GL, those in the anterior part of the GL have micropyles but no perivitelline space (are not yet fertilized), whereas those in the posterior part of the GL are fertilized. In our histological analysis, we found free sperm in the posterior area of the GL. We conclude that ovulated eggs may be self-fertilized in the posterior GL.

Development and application of an effective detection method for fish plasma vitellogenin induced by environmental estrogens

Vitellogenin is a protein induced by estrogens, including environmental chemicals with estrogenic activity. To measure the effects of environmental estogens, we developed an effective and rapid one-step method of detecting and purifying fish plasma vitellogenin using a high-performance anion-exchange chromatography column, POROS-HQ. Vitellogenin in a plasma of estradiol-treated male fish (mummichog and red sea bream) was eluted as a single peak with a retention time of 10 minutes from the column, which gives an almost pure preparation as assessed by SDS-PAGE. The lowest detectable amount of vitellogenin was 2 μg per assay. The method was used to analyze the plasma vitellogenin level of aquacultured red sea breams caught in August, when the spawning season is over, and usually no vitellogenin is detected in either females or males, physiologically. However, the data showed that in addition to a few females, some male fish synthesized vitellogenin, suggesting that some chemicals or unknown factors with estrogenic activity have induced fish in the ocean to produce vitellogenin.

The effects of 17β-estradiol on various reproductive parameters in the hermaphrodite fish Kryptolebias marmoratus.

The effect of a single injection of 17beta-estradiol (E2) was evaluated in the hermaphrodite fish Kryptolebias marmoratus. The fish [average body weight (BW), 0.15+/-0.01 g] were injected with either two concentrations of E2 (1 and 100 microg/g BW) once intraperitoneally. They were sampled at intervals of 7, 15, and 30 days after a single E2 injection. Gonadosomatic index (GSI), hepatosomatic index (HSI), the frequency of gonadal development, number of ovulated eggs, and plasma steroids levels were measured. The transcript abundances of vitellogenin (VTG) and estrogen receptors (ERalpha and beta) mRNA in the liver were also analyzed using quantitative real time polymerase chain reaction (real time PCR). GSI and the frequency of mature oocytes in the 100-microg E2-exposed group decreased compared to that of the control group during the experiment, and the number of ovulated eggs in the 100-microg E2-exposed group was lower when compared to the other groups. However, plasma E2 and 11-ketotestosterone (11-KT) levels were not significantly different between the experimental groups. On the other hand, plasma testosterone level and VTG mRNA abundance in the 100-microg E2-exposed group were significantly lower than the control group after 30 days. These results indicate that E2 stimulation at high concentration interferes with reproductive phenomena through delayed response. In addition, HSI in the 100-microg E2-exposed group and ERalpha mRNA abundance in the 1-microg E2-exposed group were significantly higher than the control group at 7 days after E2 injection, although there was no significant difference in HSI and ERalpha mRNA between all groups at 30 days. These results indicate temporal responses in reproductive parameters following high-dose E2 exposure in the hermaphrodite fish K. marmoratus.

GABA, 5-HT and amino acids in the rotifers Brachionus plicatilis and Brachionus rotundiformis.

gamma-Aminobutyric acid (GABA) and 5-hydroxytryptamine (5-HT) have been shown to increase the reproduction of the Brachionus plicatilis (NH3L strain). In the present study, the endogenous presence of GABA and 5-HT in the rotifers B. plicatilis (NH3L and Kamiura strains) and Brachionus rotundiformis (Langkawi strain) were confirmed by dot blot immunoassay and high-performance liquid chromatography (HPLC). HPLC showed that GABA and 5-HT concentrations in the three rotifer strains range from 71 to 188 pmol/mg and from 12 to 64 pmol/mg, respectively. A total of 33 amino acids were also detected in B. plicatilis and B. rotundiformis, with glutamic acid, serine, glycine, taurine, threonine, alanine, arginine, proline, valine and isoleucine in high concentrations relative to other amino acids.

Gonadal histology and serum vitellogenin levels of bigeye tuna Thunnus obesus from the Northern Pacific Ocean--absence of endocrine disruption bio-indicators.

Endocrine disrupting chemicals such as organochlorines have been detected in a large number of marine fish. Histological observation of the gonads, measurement of serum vitellogenin (VTG) level and of liver polychlorinated biphenyl (PCB) content were performed to evaluate the reproductive health and the contamination with endocrine disruptors in bigeye tuna Thunnus obesus, collected in the northern Pacific Ocean in 1999 and 2000. Abnormalities commonly found in species affected by endocrine disruptors such as the presence of oocytes in the testis or elevated serum VTG levels were not found in any of males examined. Both males and females had only small amounts of liver PCB content. The results suggest that currently there is little if any risk of organochlorine contamination or endocrine disruption of gonadal function in bigeye tuna from the northern Pacific Ocean. However, further studies are necessary to evaluate the health status of the open sea fishery resources.

Change in the transfer profile of orally administered tetrodotoxin to non-toxic cultured pufferfish Takifugu rubripes depending of its development stage.

To investigate the effects of growth (organ development) on tetrodotoxin (TTX) dynamics in the pufferfish body, TTX-containing feed homogenate was administered to 6- and 15-month old non-toxic cultured specimens of the pufferfish Takifugu rubripes at a dose of 40 mouse units (MU) (8.8 μg)/20 g body weight by oral gavage. After 24 h, the specimens were killed and the skin tissues (dorsal and ventral), muscle, liver, digestive tract, and gonads were separated. TTX content (μg/g) in each tissue, determined by liquid chromatography/mass spectrometry, revealed that the TTX distribution profile, particularly the TTX content of the liver, greatly differed between the two ages; the TTX score of 15-month old fish (3.3 μg/g) was nearly 5-fold that of 6-month old fish (0.68 μg/g). The total remaining TTX amount per individual (relative amount to the given dose) was 31% in 6-month old fish, of which 71% was in the skin, and 84% in 15-month old fish, of which 83% was in the liver. The gonadosomatic index (GSI) and hepatosomatic index (HSI) scores, and histologic observations of the gonads and liver suggest that although there is little difference in maturation stage between these two ages, there are clear distinctions in the developmental stage of the liver. The results suggest that the TTX dynamics in T. rubripes are linked to the development of the liver, i.e., the TTX taken up into the pufferfish body via food organisms is eliminated or transferred mainly to the skin in young fish with an undeveloped liver, but as the fish grow and the liver continues to develop, most of the TTX is transferred to and accumulated in the liver.