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DOI： 10.1016/j.ygcen.2022.114107

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Brill  

Dietary manipulation to maintain fish health and reduce bacterial infection through the use of immunostimulants has been widely used worldwide. A broad range of bioactive substances capable of optimising animal health has been found in several insect species, including antimicrobial/antiviral peptides, polysaccharides such as chitin, lauric acid, and insect products such as honey. Recently, we identified a novel bioactive polysaccharide from Bombyx mori, termed silkrose-BM, that can activate innate immunity in mammalian RAW264.7 macrophages and provide effective protection against vibriosis in penaeid prawns. However, the efficacy of dietary silkrose-BM in teleosts remains unclear. Here, we investigated the effects of dietary inclusion of silkrose-BM in Japanese medaka (Oryzias latipes) after they were artificially challenged with Edwardsiella tarda. The survival of medaka after infection with E. tarda was significantly improved by dietary silkrose-BM at a concentration of 10, 100, and 1000 ng/g. RNA-seq analysis was performed in the intestine and liver of the medaka to identify changes in the transcriptional profiling evoked by silkrose-BM. The dietary silkrose-BM group showed 1,194 and 2,259 differentially expressed genes (DEGs) in the intestine and liver, respectively, when compared with the control group prior to E. tarda infection. Functional enrichment analysis of DEGs showed several putative genes involved in the Toll-like receptor/nuclear factor κB pathway, cytokine-cytokine receptor interactions, complement cascade, antimicrobial peptides, and junctional modification. Taken together, these results suggest that silkrose-BM used as an immunostimulant can improve the immune system and resistance to edwardsiellosis in teleosts.

DOI： 10.3920/jiff2021.0108

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Authorship：Lead author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

The effect of silkworm-derived polysaccharide silkrose on fish ectoparasites was investigated. When juvenile yellowtail (Seriola quinqueradiata) fed diets containing silkrose were artificially infected with Benedenia seriolae, a fish ectoparasite, the numbers of parasitized B. seriolae were significantly lower compared to that in fish in the control group without silkrose treatment. Furthermore, when juvenile yellowtails were severely infected with B. seriolae, no mortality was observed in the silkrose-treated group, compared to more than 60% in the control group. In field studies carried out at a fish farm with yellowtail and white trevally (Pseudocaranx dentex), oral treatment with silkrose significantly reduced B. seriolae parasitism in yellowtail and Caligus longipedis and Neobenedenia girellae parasitism in white trevally. Silkrose treatment also reduced blood levels of cortisol, a stress hormone in both species. The changes in gene expression in the epidermis of yellowtail by silkrose treatment were also investigated, showing that the expression of various genes, including factors involved in immunity, stress response, and wound healing, was changed by the treatment. These findings indicate that silkworm-derived silkrose effectively prevents infection by external parasites in yellowtail and white trevally.

DOI： 10.3390/fishes7010014

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The term corallivorous gastropod refers to a group of snails that feeds on coral and inhabits coral communities worldwide. Outbreaks of these species cause serious damage to coral communities. There are various reasons behind the outbreaks; however, further clarifications are needed. It may be possible to predict outbreaks by measuring the number of floating larvae of corallivorous gastropods in seawater. Drupella fragum is the most damaging species in Japan, so we produced antibodies against D. fragum larvae in order to easily detect this species in the field. Antibody specificity analysis in aquarium-hatched corallivorous gastropods showed a higher specificity against D. fragum compared to D. cornus. A field study using the antibody showed that many D. fragum larvae were detected from June to November at all stations. The larvae at the Shirigai station were collected in June and July in large numbers compared to the other stations. Large groups of D. fragum were collected around the sampling point in Shirigai in September 2016. Our results imply that there is a possibility that outbreaks could be predicted using this antibody.</p>

DOI： 10.3390/su132111713

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Against a background of increased demand for fish meal (FM), black soldier fly larva is a promising alternative feed source for sustainable aquaculture. Yellowtail, the most popular farmed fish in Japan, is a carnivorous fish; therefore, it requires a high proportion of FM in its diet. This study represents the first example of yellowtail fed on a diet including insect meal as a replacement for FM. Partially defatted black soldier fly meal (PDBM) comprised 49.0% crude protein and 23.2% crude fat, while completely defatted black soldier fly meal (CDBM) contained less than 10% crude fat, as the same level as FM was achieved with defatting PDBM using hexane. In feeding trials, growth of the fish was reduced in accordance with PDBM content: 10%, 20%, and 30% in their diet. Although a diet including 8% CDBM (with the same protein composition as 10% PDBM) also resulted in decreased fish growth, growth with a diet including 16% CDBM (with the same protein composition as 20% PDBM) was significantly higher than that of 20% PDBM, and equivalent to that of 10% PDBM. Therefore, even 10% of partially or completely black soldier fly larvae meal in diets inhibited growth in juvenile yellowtail, and we found that removal of the fat fraction could improve fish growth.

DOI： 10.3390/insects12080722

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

We previously identified a novel acidic polysaccharide, silkrose-AY, from the Japanese oak silkmoth (Antheraea yamamai), which can activate an innate immune response in mouse macrophage cells. However, innate immune responses stimulated by silkrose-AY in teleosts remain unclear. Here, we show the influence of dietary silkrose-AY in medaka (Oryzias latipes), a teleost model, in response to Edwardsiella tarda infection. Dietary silkrose-AY significantly improved the survival of fish and decreased the number of bacteria in their kidneys after the fish were artificially infected with E. tarda by immersion. We also performed a microarray analysis of the intestine, which serves as a primary barrier against microbial infection, to understand the profiles of differentially expressed genes (DEGs) evoked by silkrose-AY. The dietary silkrose-AY group showed differential expression of 2930 genes when compared with the control group prior to E. tarda infection. Gene ontology and pathway analysis of the DEGs highlighted several putative genes involved in pathogen attachment/recognition, the complement and coagulation cascade, antimicrobial peptides/enzymes, opsonization/phagocytosis, and epithelial junctional modification. Our findings thus provide fundamental information to help understand the molecular mechanism of bacterial protection offered by insect-derived immunostimulatory polysaccharides in teleosts.

DOI： 10.1016/j.fsi.2021.05.001

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.aquaculture.2019.734667

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI  

In our study, a novel bioactive polysaccharide was identified in the larvae of the black soldier fly (BSF) (Hermetia illucens) as a molecule that activates the mammalian innate immune response. We attempted to isolate this molecule, which was named dipterose-BSF, by gel-filtration and anion-exchange chromatography, followed by nitric oxide (NO) production in mouse RAW264.7 macrophage cells as a marker of immunomodulatory activity. Dipterose-BSF had an average molecular weight of 1.47 × 105 and consisted of ten monosaccharides. Furthermore, in vitro assays demonstrated that dipterose-BSF enhanced the expression of proinflammatory cytokines and interferon β (IFNβ) in RAW264.7 cells. The inhibition of Toll-like receptor 2 (TLR2) and 4 (TLR4) significantly attenuated NO production by dipterose-BSF, indicating that dipterose-BSF stimulates the induction of various cytokines in macrophages via the TLR signaling pathway. This observation was analogous with the activation of nuclear factor kappa B in RAW264.7 cells after exposure to dipterose-BSF. Our results suggest that dipterose-BSF has immunomodulatory potential through activating the host innate immune system, which allows it to be a novel immunomodulator for implementation as a functional food supplement in poultry, livestock, and farmed fish.

DOI： 10.3390/biom9110677

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© 2019 Vertebral deformities, including kyphosis, of the cultured yellowtail Seriola quinqueradiata resulted in a decrease in product value, and consequently economic loss for fish farmers. Hence, in order to resolve this problem, it is important to examine the characteristics of kyphotic vertebrae in fish. In this study, we focused on the comparison between kyphotic and non-kyphotic two-year old yellowtail. We analyzed the appearance of the caudal vertebrae by X-ray image analysis. The strength of the vertebrae, bone density and gonad development were analyzed. Moreover, the muscles were analyzed for protein, fat, moisture, and ash content. A decrease in vertebrae strength and vertebrae bone density, thinning of compact bone, and an increase in bone porosity relative to the total spongy bone area of vertebrae were observed in kyphotic fish. However, there is no difference in weights, and there is no effect on the fleshy substance component of muscles. Histological observation of gonad suggested that spermatogenesis progressed in kyphotic fish. Our study implies that a thin compact bone area and an increase in bone porosity caused low bone strength and density, and weak vertebrae in kyphotic fish.

DOI： 10.1016/j.aquaculture.2019.03.071

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Black soldier fly (BSF) larvae and pre-pupae could be satisfactorily raised on household organic waste and used as poultry feed, offering a potential sustainable way to recycle untapped resources of waste. The present study was conducted to determine if whole (non-defatted) BSF larvae and pre-pupae raised on experimental household waste could substitute soybean meal and oil as ingredients for laying hen diets. While no significant differences in feed intake and the egg-laying rate of hens were observed throughout the experiment, egg weight and eggshell thickness were greater in the pre-pupae-fed group than in the other groups. Moreover, although diversity of the cecal microbiota was significantly higher in the pre-pupae-fed than in the control group, no significant differences in bacterial genera known to cause food poisoning were observed when comparing the treatment groups. Nonetheless, Lactobacillus and Bifidobacterium populations were significantly lower in the treatment than in the control group. Fat content in BSF was possibly related with the changes in the cecal microbiota. Hence, since BSF fat was deficient in essential fatty acids, special attention should be paid to the fat content and its fatty acid composition in the case of regular inclusion of BSF larvae and pre-pupae oil as an ingredient in poultry diets.

DOI： 10.3390/ani9030098

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI  

Yellow mealworm (Tenebrio molitor) larvae are a potential alternative animal protein source for sustainable aquaculture. However, reports on the successful complete substitution of fish meal with yellow mealworm larvae in an aquaculture diet have been limited. In this study, we conducted a feeding trial with red seabream (Pagrus major) being fed diets with partial or complete replacement of fish meal with yellow mealworm larvae defatted with a hexane⁻ethanol solution. Feed intake in red seabream increased in accordance with yellow mealworm larvae inclusion, and diets including 65% defatted mealworm larvae (complete replacement of fish meal) showed significant growth promotion. The addition of the oil fraction from mealworm larvae to diets resulted in growth reduction, despite meeting the nutritional requirements of red seabream. Moreover, the survival rate of red seabreams fed diets with partial replacement of fish meal with mealworm larvae was significantly higher in a challenge test with pathogenic Edwardsiella tarda bacteria. The present study demonstrated that yellow mealworm larvae are not merely an alternative animal protein, but have potential as functional feed ingredients for aquaculture production.

DOI： 10.3390/ani9030100

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© 2019 Elsevier Inc. Receptors for follicle-stimulating hormone (Fshr), luteinizing hormone (Lhcgr1 and Lhcgr2) and androgens (Ara and Arb) transduce the hormonal signals that coordinate spermatogenesis, but the factors that regulate the abundance of these transducers in fish testes remain little-understood. To mend this paucity of information, we first determined changes in transcript abundance for these receptors (fshr, lhcgr1, ara and arb) during spermatogenesis induced by human chorionic gonadotropin (hCG) injection in the eel, Anguilla australis. We related our findings to testicular production of the fish androgen, 11-ketotestosterone (11-KT), and to the levels of the transcripts encoding steroidogenic acute regulatory protein (star) and 11β-hydroxylase (cyp11b), and subsequently evaluated the effects of hCG or 11-KT on mRNA levels of these target genes in vitro. Testicular 11-KT production was greatly increased by hCG treatment, both in vivo and in vitro, and associated with up-regulation of star and cyp11b transcripts. In situ hybridization indicated that testicular fshr mRNA levels were higher in the early stages of hCG-induced spermatogenesis, while lhcgr1 transcripts were most abundant later, once spermatids were observed. In vitro experiments further showed that hCG and its steroidal mediator 11-KT significantly increased fshr transcript abundance. These data provide new angles on the interactions between gonadotropin and androgen signaling during early spermatogenesis. Increases in levels of 11-KT following hCG injection elevated testicular fshr mRNA levels augmenting Fsh sensitivity in the testis. This evidence is suggestive of a positive feedback loop between gonadotropins and 11-KT that may be key to regulating early spermatogenesis in fish.

DOI： 10.1016/j.ygcen.2019.04.020

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI  

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. Insects are an attractive alternative to fish meal (FM) as a sustainable protein source in aquaculture feed that does not negatively impact the marine ecosystem. Despite housefly (Musca domestica) larvae having adequacy of amino acid profiles, they have sometimes been reported to be inferior to FM, especially for marine carnivorous fish species. Here, we report that the removal of the hydrophobic fractions from housefly larvae enables significant replacement of FM in the diet of the red seabream (Pagrus major). In a feeding trial, housefly (HF) larvae that had the hydrophobic fraction removed as a complete substitution for 70% FM produced satisfactory growth. However, HF larvae that were supplemented with the hydrophobic fraction resulted in significant growth reduction. Growth recovery was incomplete by supplementation of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) to undefatted HF larvae, being equivalent to that of fatty acid content with a control diet. Moreover, fish with a dietary intake of catechol identified from the hydrophobic fraction of the HF showed growth reduction and morphological alterations in the intestine. Our findings indicate that the hydrophobic fraction from HF larvae contains a negative factor for fish growth and eliminating the fraction from HF larvae is thought to be an important process for sustainable aquaculture.

DOI： 10.3390/fishes4030038

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

We previously identified novel bioactive polysaccharides from Bactrocera cucurbitae and Antheraea yamamai that activate innate immunity in RAW264 murine macrophages. However, in terms of potential applications in the cultivation of prawns, there were problems with the availability of these insects. However, we have now identified a polysaccharide from Bombyx mori that activates innate immunity in RAW264 cells and penaeid prawns. This purified polysaccharide, termed silkrose of B. mori (silkrose-BM), has a molecular weight of 1,150,000 and produces a single symmetrical peak on HPLC. Eight of nine constitutive monosaccharides of silkrose-BM are concomitant with dipterose of B. cucurbitae (dipterose-BC) and silkrose of A. yamamai (silkrose-AY). The major differences are found in the molar ratios of the monosaccharides. Silkrose-BM is approximately 500-fold less potent than silkrose-AY (EC50: 2.5 and 0.0043 μg/mL, respectively) in a nitrite oxide (NO) production assay using RAW264 cells. However, the maximum NO production for silkrose-BM and AY were comparable and higher than that of the lipopolysaccharide of Escherichia coli. The survival of penaeid prawns (Litopenaeus vannamei and Marsupenaeus japonicus) after infection with Vibrio penaecida was significantly improved by both dietary silkrose-BM and B. mori pupae. This suggests that silkrose-BM effectively prevents vibriosis in penaeid prawns via the activation of innate immunity.

DOI： 10.1038/s41598-018-27241-3

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BioMed Central Ltd.  

Background: Pearl production by transplantation in Akoya pearl oyster (Pinctada fucata) is a biotechnology developed in Japan that skillfully utilizes the pearl-forming ability of oysters. In this method, cultured pearls are formed from a pearl nucleus and a small piece of mantle transplanted into the gonads of recipient pearl oysters. In this study, we hypothesized that the sex of the recipient pearl oyster might affect the quality of pearl produced. While some previous studies have examined the sex of Akoya pearl oyster, detailed information is lacking. Results: To investigate sex in Akoya pearl oyster, we collected small gonadal fragments from 1-year-old pearl oysters by biopsy. Using the collected gonad fragment, the sex of the oysters was determined by microscopic observation, and the remaining samples were stored for gene expression analyses. All oysters were labeled to distinguish each individual for serial samplings every four months over the 2-year study period. At the start of experiment, nearly all of the pearl oysters were male, but the male:female ratio ofmale decreased over the course of the experiment. Interestingly, the number of males increased after spring, during the breeding season. This suggests that, in pearl oyster, sex is affected by season. Expression analysis of sex-related genes (Dmrt2, Vtg, Zp) indicated that all genes were expressed in all individuals and all periods. Conclusions: These results suggest that Akoya pearl oysters are hermaphroditic, and that females appear as necessary, such as during the breeding season. These findings could contribute to higher efficiency and quality of pearl cultivation.

DOI： 10.1186/s40851-018-0098-7

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The improvement in feed efficiency is one of the most important subjects in fish culture. The development of feed, in terms of good intake, high growth performance, and high feed efficiency is needed. Squid viscera are one of the candidates for alternative material in improving feed efficiency in fish culture. In the present study, we described the dietary effect of the squid viscera hydrolysate (SVH) on the growth performance of the red sea bream. The addition of SVH to feed caused significant increases in feed intake, fork length, and body weight and produced a marked improvement in feed conversion after 4 weeks of feeding. Furthermore, the results of this feeding revealed that low dietary levels of SVH promote growth performance in the red sea bream. We physiologically analyzed digestion and appetite in fish fed diet containing SVH. SVH promoted the activity of hepatic trypsin and lipase, gene expression of stomach pepsin, hepatic lipase, and pyloric caeca trypsin, thereby improving the nutrient availability in red sea bream. Moreover, the mRNA expression of appetite regulating factor, such as brain NPY and stomach ghrelin was significantly improved by dietary SVH. Our current results indicate that dietary SVH as alternative material produced excellent effects on growth performance, which is dependent on the promoting effect on digestion and appetite in red sea bream.

DOI： 10.1007/s10695-017-0391-y

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Bioremediation is a promising method for remediating environmentally polluted water. We investigated the abilities of two benthic annelid species to biotransform 1-nitronaphthalene, a nitrated polycyclic aromatic hydrocarbon. We used an oligochaete, Thalassodrilides sp. (Naididae), collected from the sediment beneath a fish farm and a polychaete, Perinereis nuntia, which was obtained from a commercial source. Populations of both organisms were exposed to 1400 mu g L-1 of 1-nitronaphthalene in seawater for 3 days in the dark at 20 degrees C. The concentration of the pollutant decreased to 12 mu g L-1 in the seawater containing the Thalassodrilides sp. and to 560 mu g L-1 in the seawater containing P. nuntia. The 1-nitronaphthalene concentration in the bodies of the animals increased from 12 to 94 mu g kg(-1) in Thalassodrilides sp. and from 0.90 mu g kg(-1) to 38,000 mu g kg(-1) in P. nuntia. After 3 days, 99% and 40% of the 1-nitronaphthalene had been biotransformed in the Thalassodrilides sp. and P. nuntia experimental groups, respectively. We then tested the acute toxicity of residual 1-nitronaphthalene from the same water using mummichog (fish) larvae. After the larvae had been exposed for 96 h, the percentage of apparently unaffected larvae remaining was 83.3% in Thalassodrilides sp. group but only 16.7% in the P. nuntia group. Clearly, of the two species we studied, Thalassodrilides sp. had a superior ability to convert 1-nitronaphthalene into substances that were nontoxic to mummichog larvae. Therefore, we recommend the use of this species for bioremediation of chemically polluted sediments. (C) 2016 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.chemosphere.2016.02.026

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We have identified a novel acidic polysaccharide from silkmoth (Antheraea yamamai) pupae that activates the mammalian innate immune response. This bioactive polysaccharide was isolated using nitric oxide production in mouse RAW264 macrophages as an indicator of immunostimulatory activity. We named this polysaccharide "silkrose". It has a molecular weight of 3.15 x 10(5) and comprises nine monosaccharides. The expression profiles indicated that silkrose induced the expression of proinflammatory cytokines and interferon 13 that exist downstream of MyD88-dependent and MyD88-indeptendent signaling pathways. Also, the inhibition of Toll-like receptor 4 (TLR4), which exists upstream of the signaling pathways, led to the suppression of NO production by silkrose. Furthermore, this polysaccharide promoted the activation of nuclear factor kappa B in RAW264 cells, indicating that it stimulates the induction of various cytokines in macrophages through the TLR4 signaling pathway. Our results thus suggest that silkrose activates the innate immune response to various pathogenic microorganisms and viral infections. (C) 2015 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.carbpol.2015.09.070

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC FISHERIES SCIENCE  

To develop an extruded pellet (EP) appropriate for the cultured bluefin tuna Thunnus orientalis, we compared the feeding effects of EP and raw fish feed on their growth and digestion. EP-fed tuna had slower growth compared to raw fish-fed tuna after a decrease in pepsin activity, and growth was restored to the same level as that of the control when protease activity increased resulting from a bloated pyloric caeca. EP feeding delayed the intestinal arrival time of food compared to raw fish feeding, because food is retained for longer in the stomach. This corresponded with the time of expression of digestive-related factors in both groups, resulting in delayed expression of digestive enhancer (cck) and digestive suppressor (pyy) in the EP group compared with the control group. The expression levels of Ghrelin, a growth related factor expressed when nutrients are absorbed into the intestine, were also significantly higher in the EP group than in the control group. Moreover, expression levels and times of growth hormone (gh) were not significantly different between EP and raw fish feed. Therefore, bluefin tuna has the capacity to adapt their digestive physiology in response to EP in order to grow efficiently.

DOI： 10.2331/suisan.16-0030

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC FISHERIES SCIENCE  

To develop an extruded pellet (EP) appropriate for the cultured bluefin tuna Thunnus orientalis, we compared the feeding effects of EP and raw fish feed on their growth and digestion. EP-fed tuna had slower growth compared to raw fish-fed tuna after a decrease in pepsin activity, and growth was restored to the same level as that of the control when protease activity increased resulting from a bloated pyloric caeca. EP feeding delayed the intestinal arrival time of food compared to raw fish feeding, because food is retained for longer in the stomach. This corresponded with the time of expression of digestive-related factors in both groups, resulting in delayed expression of digestive enhancer (cck) and digestive suppressor (pyy) in the EP group compared with the control group. The expression levels of Ghrelin, a growth related factor expressed when nutrients are absorbed into the intestine, were also significantly higher in the EP group than in the control group. Moreover, expression levels and times of growth hormone (gh) were not significantly different between EP and raw fish feed. Therefore, bluefin tuna has the capacity to adapt their digestive physiology in response to EP in order to grow efficiently.

DOI： 10.2331/suisan.16-0030

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Insects are attracting increasing worldwide attention as living organisms that produce various useful substances. Although a wide variety of bioactive substances have been identified in insects, there has been little study of the dietary effects of insects that contain these useful substances. In our present study, we describe the dietary effects of housefly [Musca domestica (Linnaeus)] pupae in the red sea bream [Pagrus major (Temminck and Schlegel)]. The addition of low levels of housefly pupae to the diets of these fish caused significant increases in fork length and body weight and produced a marked improvement in feed conversion after 24 days of feeding. Furthermore, the results of 6-month feeding trials revealed that low dietary levels of housefly pupae promote growth performance in the red sea bream. We further found that dietary housefly pupae significantly enhance the peritoneal leukocyte phagocytic activity in the red sea bream and provides increased protection against a lethal challenge from a major pathogen in this species, Edwardsiella tarda (Ewing et al). We also show that housefly pupae dietary supplements do not negatively affect the health of rats [Rattus norvegicus (Berkenhout)]. Our current results indicate the great potential of insects as functional feed sources for farmed fish.

DOI： 10.1007/s13355-015-0325-z

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During the cultivation of pearls, a mantle piece and pearl nucleus are transplanted into a recipient Akoya pearl oyster (Pinctada fucata). The gonad has been the area of transplantation since pearl culture techniques were originally developed. Transplantation into gonads without germ cells is vital for the production of high-quality pearls. Almost all pearl cultivators thus perform forced elimination of the germ cells from the gonads before transplantation. However, little attention has been paid to the sex of the pearl oyster, even though the gonad state is vital for the production of high-quality pearls. In our present study, we investigated the relationship between the sex of the recipient Akoya pearl oyster and pearl quality. We found that the rate of production of commercially valuable pearls was higher for male than for female recipient oysters. Furthermore, the nacre in male recipient oysters grew evenly everymonth whereas it was added less uniformly in female recipient oysters. Nacre growth in female pearl oysters was found to be related to ovarian development. These results indicate that differences exist in the pearl formation ability of male and female Akoya pearl oysters. By understanding the relationship between the sex of Akoya pearl oysters and pearl quality, high-quality pearls could be cultivated with better efficiency. (C) 2014 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.aquaculture.2014.12.028

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A novel water-soluble polysaccharide was identified in the pupae of the melon fly Bactrocera cucurbitae) as a molecule that activates the mammalian innate immune response. We attempted to purify this innate immune activator using nitric oxide NO) production in mouse RAW264 macrophages as an indicator of immunostimulatory activity. A novel acidic polysaccharide was identified, which we named "dipterose'', with a molecular weight of 1.01 x 10(6) and comprising nine monosaccharides. Dipterose was synthesized in the melon fly itself at the pupal stage. The NO-producing activity of dipterose was approximately equal to that of lipopolysaccharide, a potent immunostimulator. Inhibition of Toll-like receptor 4 TLR4) led to the suppression of NO production by dipterose. Furthermore, dipterose induced the expression of proinflammatory cytokines and interferon beta (IFN beta) and promoted the activation of nuclear factor kappa B (NF-kappa B) in macrophages, indicating that it stimulates the induction of various cytokines in RAW264 cells via the TLR4 signaling pathway. Our results thus suggest that dipterose activates the innate immune response against various pathogenic microorganisms and viral infections. This is the first identification of an innate immune-activating polysaccharide from an animal.

DOI： 10.1371/journal.pone.0114823

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Growth initiates sexual maturation in fish but the onset of maturity also leads to a marked loss in body weight. To avoid the growth loss associated with spawning, yellowtail (Seriola quinqueradiata) farmers have attempted to restrict the diets of their fish stocks from winter to spring, the period just before the breeding season. This prevents reductions in body weight. In our present study, we show through endocrinological and physiological analyses of harm-cultured yellowtails that the growth of these fish can be regulated through dietary control. Our results indicate that the body weights of a diet restricted yellowtail group gradually increases through the breeding season compared with the controls. Both the body weights and folk lengths of the diet restricted group surpassed the control groups. In the spawning season, the diet restricted group had smaller gonads than the controls in both sexes. However, the gonad somatic index (GSI) rapidly increased in the control group compared with the restricted group and histological observations of the controls further indicated that the testes contained sperm, and that the female gonads contained numerous oocytes that had accumulated yolk. These findings confirm that a restriction of the diet suppresses gonad development during the yellowtail breeding season. The sex steroid hormone, 17 alpha, 20 beta-diOHprog, was found to be associated with this dietary control of gametogenesis as it showed decreased levels in the restricted groups compared with the controls. To further investigate the functions of 17 alpha, 20 beta-diOHprog on fish growth, this hormone was administered intraperitoneally to juvenile yellowtails. Implanted fish showed a remarkable increase in body weight compared with the control group and the lipase activities of the dorsal and ventral muscles of 17 alpha, 20 beta-diOHprog-injected fish were also higher than those in the control group. We conclude from our data that the regulation of the yellowtail diet can control their growth and gametogenesis. (C) 2014 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.aquaculture.2013.12.031

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The brightness and color of pearls varies among different pearl-producing shellfish and have been a source of human fascination since ancient times. When produced through cultivation, the characteristics and quality of a pearl depend on the kind of shellfish used and also the transplanted mantle graft. This suggests that the Akoya pearl oyster, which is generally used in Japan for pearl culturing, can produce different kinds of pearl through the use of mantles from different species of shellfish. However, a transplanted heterogeneous mantle would be rejected by the immune system of the Akoya oyster. We have therefore developed a new method to suppress the Akoya immune system that archives immune tolerance to other shellfish. It is generally known that small quantities of antigens can be used to produce archived immunological tolerance in a clinical setting. We successfully suppressed the Akoya pearl oyster immune response against a Mab, pearl oyster graft through repeat injections of mantle homogenates. We then transplanted a Mab, pearl oyster mantle graft into the immunologically tolerant Akoya pearl oyster and obtained a Mab, pearl from an Akoya pearl oyster. Our new technique thus makes the production of novel and different pearls in the Akoya possible. We believe that this has significant future potential for the advancement of the pearl industry.

DOI： 10.1007/s10126-013-9525-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Earthworms ingest various materials in addition to food items, such as soil particles. Most earthworms of the family Megascolecidae, a dominant family in Japan, have intestinal caeca connected directly to the intestinal tract. The function of the caeca has not been demonstrated, although it is thought to be associated with digestion. We investigated the activity of the digestive enzymes amylase, phosphatase, cellulase, and protease in different regions of the gut, including the intestinal caeca, in three species of megascolecid earthworms, Pheretima heteropoda, Pheretima hilgendorfi, and Pheretima sieboldi. Activities of several enzymes were high in the intestinal caeca; in particular, protease activity was higher in the caeca than that in the anterior gut, foregut, midgut, and hindgut in all three species. Moreover, the ratio of enzyme activities in the intestinal caeca to whole-gut tended to be higher in manicate intestinal caeca than in simple intestinal caeca. These results suggest that the digestive system of earthworms relies on the intestinal caeca.

DOI： 10.2108/zsj.30.710

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

Meiosis is a unique and critical process in reproduction. Although the key molecular components of meiosis have been identified, the molecular mechanisms regulating the entry into this pathway remain unclear. We previously demonstrated that a progestin in teleost fish, 17alpha, 20beta-dihydroxy-4-pregnen-3-one, is essential for meiotic initiation, and up-regulates taurine synthesis and the production of trypsin in Sertoli cells. In the present study, we found that trypsin promotes the uptake of taurine into germ cells through the up-regulation of solute carrier family 6 (neurotransmitter transporter, taurine), member 6 (Slc6a6) expression. We further found that this up-regulation of the taurine signal is required for Spo11a expression and meiotic initiation.

DOI： 10.1095/biolreprod.113.109777

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER WIEN  

It has been demonstrated that taurine has various physiological functions in the body. We demonstrated that taurine is abundant in the serum, liver, muscle and testis of the Japanese eel (Anguilla japonica). In the eel testis, taurine is found mainly in spermatogonia and is weakly expressed also in the Sertoli cells. We have further found in the eel testis that taurine is actively accumulated via the sodium/chloride-dependent taurine transporter (TauT; SLC6A6), which is expressed in germ cells. In our current study, the effects of taurine on the anti-oxidant response were examined. Taurine was found to promote the total superoxide dismutase (SOD) activity in the testis. Moreover, our results indicate that taurine does not affect the mRNA levels of copper-zinc (Cu/Zn) SOD or manganese SOD, but promotes the translation of Cu/Zn SOD. Overall, our present data suggest that taurine may modulate Cu/Zn SOD at the translational level and thereby may play an important role in the protection of germ cells from oxidative stress.

DOI： 10.1007/s00726-012-1316-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

Oxidative stress has been implicated in pathogenesis of many diseases, but few studies describe its influence on spermatogenesis. In this study, we analyzed the direct influence of hypoxanthine (Hx)-induced reactive oxygen species (ROS) on spermatogenesis in fish using the Japanese eel (Anguilla japonica) testicular organ culture system. Testicular fragments of eels were cultured in 0.1-100 mu M Hx with or without 10 ng/ml 11-ketotestosterone (11-KT). Immunohistochemistry for 5-bromo-2-deoxyuridine showed that Hx treatment at a low dose (1 mu M) already inhibits 11-KT-induced germ cell proliferation after culture. An in situ TUNEL assay and 8-hydroxy-2'-deoxyguanosine immunohistochemistry revealed an intense germ cell apoptosis and high oxidative DNA damage in testicular fragments cultured at the highest dose of Hx (100 mu M) with 11-KT. A total superoxide dismutase (SOD) activity assay showed a decrease in SOD activity in testicular fragments cultured with 11-KT. These data suggest that ROS may directly inhibit spermatogenesis, and that decreased SOD activity renders proliferating spermatogonia susceptible to ROS, hence leading to apoptosis.

DOI： 10.1095/biolreprod.112.099887

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

To develop an efficient and stable method for enhancing spawning of black scrapers in captivity, we examined the following three factors: (1) different kinds of spawning substrate, (2) the area of the spawning substrate, (3) the grain size of the spawning substrate. The results demonstrated that sand is the optimum spawning substrate when compared to a hard polyvinyl chloride sheet, a piece of shading net, or artificial spawning grass. No difference was observed in the spawning frequency between a small sand bed of area 400 cm(2) and sand spread over the bottom of the tank to an area of 7085 cm(2), since the egg masses were spawned on the sand in a small area approximately 15 cm in diameter. In addition to the spawned eggs, the small movable sand bed is useful for subsequent harvest. No differences were observed in either the spawning frequency or the hatching rate when using sand beds with different grain sizes. In large-scale egg collection trials, an average of 33 egg masses from which an average of 403,000 larval fish were obtained. We can therefore conclude that the procedure developed in the present study is applicable to the commercial-scale seed production of black scraper.

DOI： 10.1007/s12562-012-0527-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER WIEN  

In teleost fish, the progestin 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP) is an essential component of the spermatogenesis pathway. In a series of investigations on the mechanisms underlying progestin-stimulated spermatogenesis, we have found that DHP up-regulates the expression of cysteine dioxygenase1 (CDO1) in the Japanese eel testis. CDO1 is one of the enzymes involved in the taurine biosynthesis pathway. To evaluate whether taurine is synthesized in the eel testis, cysteine sulfinate decarboxylase (CSD), another enzyme involved in taurine synthesis, was isolated from this species. RT-PCR and in vitro eel testicular culture revealed that although CSD was also expressed in eel testis, neither DHP nor other sex steroids affect CSD mRNA expression in a similar manner to CDO1. Using an in vitro eel testicular culture system, we further investigated the effects of DHP on taurine synthesis in the eel testis. HPLC analysis showed that DHP treatment significantly increases the taurine levels in the eel testis. These results suggest that DHP promotes taurine synthesis via the up-regulation of CDO1 mRNA expression during eel spermatogenesis. Furthermore, we observed from our analysis that although taurine does not induce complete spermatogenesis, it promotes spermatogonial DNA synthesis and the expression of Spo11, a meiosis-specific marker. These data thus suggest that taurine augments the effects of sex steroids in the promotion of spermatogonial proliferation and/or meiosis and hence that taurine plays important roles in spermatogenesis.

DOI： 10.1007/s00726-011-1128-3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

In general, there is a relationship between growth and reproduction, and gonads are known to be important organs for growth, but direct evidence for their role is lacking. Here, using a fish model, we report direct evidence that gonads are endocrine organs equal to the pituitary in controlling body growth. Gonadal loss of function, gain of function, and rescue of growth were investigated in tilapia. Gonadectomy experiments were carried out in juvenile males and females. Gonadectomy significantly retarded growth compared with controls; however, this retardation was rescued by the implantation of extirpated gonads. Because gonads express growth hormone, it is possible that gonads control body growth through the secretion of growth hormone and/or other endocrine factors. We propose that gonads are integral players in the dynamic regulation of growth in teleosts.

DOI： 10.1073/pnas.1118704109

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

A luteinizing hormone receptor (lhr) cDNA with high identity to other fish lhrs was fully cloned from the ovary of the Japanese eel (Anguilla japonica). The genes for two gonadotropin receptors (Gthr), follicle-stimulating hormone receptor (fshr) and lhr, were differentially expressed during oogenesis, which was artificially induced by salmon pituitary extract, a gonadotropin-rich source. Transcript abundance of fshr was significantly elevated at the early vitellogenic stage and peaked at the late vitellogenic stage, while lhr gene expression rapidly induced at the late vitellogenic stage and thereafter remained at a high level. The abundance of fshr and lhr transcripts was highest in the ovary in female eels. In addition to the ovary, forebrain was a major site for the fshr transcript, although the level did not change with reproductive status. Furthermore, it was examined how eel Gthrs were activated by two mammalian chrionic gonadtropin (CG), equine CG (eCG) and human CG (hCG), that have been used for study of fish reproduction as substitutes for homologous Gths. Both CGs specifically activated eel Lhr, but not Fshr, although the degree of effectiveness was different; thus the concentration of hCG (0.1 ng/ml) required for significant activation of Lhr was much lower than that of eCG (100 ng/ml). These data on gene expression and ligand-activation of Gthrs suggest that Fsh and Lh act differentially in the regulation of reproductive function in Japanese eel.

DOI： 10.2108/zsj.29.204

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOSCIENTIFICA LTD  

Gh plays important roles in development, somatic growth and gametogenesis in vertebrates. To determine the physiological role of Gh in reproduction in male teleosts, the expression of genes encoding Gh and the two Gh receptors (Ghrs) during spermatogenesis, and the action of Gh in vitro was examined using the Japanese eel (Anguilla japonica). gh, ghr1 and ghr2 mRNA transcripts were detected in all spermatogenic stages. In situ hybridization showed the presence of ghr1 and ghr2 mRNA in the germ cells. Immunohistochemistry using an antiserum against eel Gh indicated that Gh protein was localized to Sertoli cells surrounding the germ cells in early spermatogenesis. Recombinant eel Gh induced spermatogonial proliferation in a testis organ culture system, an effect that was independent from the production of steroid hormones or Igf1. This study identifies a role for eel Gh in the regulation of early spermatogenesis, particularly in the mitotic phase of spermatogenesis, that is not mediated by either steroid hormones or Igf1 production. Reproduction (2011) 142 869-877

DOI： 10.1530/REP-11-0203

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER HEIDELBERG  

Abstract Polychaetes constitute most of the benthic macroinvertebrates in estuarine and coastal environments. We investigated the utilization of organic matter in two polychaete species, Capitella sp. I and Perinereis nuntia brevicirris, living in different coastal habitats. The protease activity of Capitella sp. I (89.7 mu g mg(-1)) was about 10 times that of P. nuntia brevicirris (8.0 mu g mg(-1)). High cellulase (endo-beta-1,4-glucanase) activity was detected in P. nuntia brevicirris (3.2 mu g mg(-1)), whereas scarcely any was detected in Capitella sp. I. We isolated cDNA clones of protease mRNA from Capitella sp. I and of cellulase mRNA from P. nuntia brevicirris. The high protease activity of Capitella sp. I enabled it to survive in the sediment under a fish farm, where it degrades organic matter. In contrast, the high cellulase activity of the estuarydwelling P. nuntia brevicirris allowed it to degrade organic matter originating from terrestrial areas.

DOI： 10.1007/s00227-011-1641-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

Interferon (IFN) plays crucial roles in innate immune responses against viral infections. In the present study, we report cloning and characterization of the IFN gene from the sevenband grouper (Epinephelus septemfasciatus), and the anti-viral effects of its recombinant IFN protein in vivo. The isolated cDNA from sevenband grouper IFN encoded a protein consisting of 178 amino acids, and its first 22 amino acids represented a putative signal peptide. We named the identified sevenband grouper IFN gene as SgIFNa1 based on the result from phylogenetic analysis that categorized the deduced protein sequence into fish IFNa family. The expression of SgIFNa1 mRNA in the head kidney cells was induced by synthetic Poly(1:C), which is known as an inducer of IFN. It has also been confirmed that injection of recombinant SgIFNa1 protein (rSgIFNa1) upregulates expression of the Mx gene, which is known as an IFN-responsive gene, in head kidney cells. Moreover, we observed that preliminarily injection of rSgIFNa1 provided significant protection against a lethal challenge of nervous necrosis virus (NNV), which is a serious disease of sevenband grouper. These results demonstrate that SgIFNa1 has anti-viral activity and the administration of rSgIFNa1 to sevenband grouper is effective in preventing severe symptom development after NNV infection. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.fsi.2011.02.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Background: Spermatogonia are highly tolerant to reactive oxygen species (ROS) attack while advanced-stage germ cells such as spermatozoa are much more susceptible, but the precise reason for this variation in ROS tolerance remains unknown.
Methodology/Principal Findings: Using the Japanese eel testicular culture system that enables a complete spermatogenesis in vitro, we report that advanced-stage germ cells undergo intense apoptosis and exhibit strong signal for 8-hydroxy-29-deoxyguanosine, an oxidative DNA damage marker, upon exposure to hypoxanthine-generated ROS while spermatogonia remain unaltered. Activity assay of antioxidant enzyme, superoxide dismutase (SOD) and Western blot analysis using an anti-Copper/Zinc (Cu/Zn) SOD antibody showed a high SOD activity and Cu/Zn SOD protein concentration during early spermatogenesis. Immunohistochemistry showed a strong expression for Cu/Zn SOD in spermatogonia but weak expression in advanced-stage germ cells. Zn deficiency reduced activity of the recombinant eel Cu/Zn SOD protein. Cu/Zn SOD siRNA decreased Cu/Zn SOD expression in spermatogonia and led to increased oxidative damage.
Conclusions/Significance: These data indicate that the presence of high levels of Cu/Zn SOD and Zn render spermatogonia resistant to ROS, and consequently protected from oxidative stress. These findings provide the biochemical basis for the high tolerance of spermatogonia to oxidative stress.

DOI： 10.1371/journal.pone.0016938

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Spermatogenesis is a developmental process during which a small number of diploid spermatogonial stem cells produce a large number of highly differentiated spermatozoa carrying a haploid, recombined genome. We characterise morphologically the different germ cell stages with particular attention for the spermatogonial generations, including the stem cells and their specific capacity to colonise a recipient&apos;s testis after transplantation. We propose a nomenclature for fish germ cells to improve the comparability among different teleost fish but also to higher vertebrates. Survival and development of germ cells depends on their continuous and close contact to Sertoli cells, and we review their multiple roles in the cystic mode of spermatogenesis seen in fish. We then discuss gene expression patterns associated with testis maturation. The endocrine system of vertebrates has evolved as master control system over spermatogenesis. In fish, both pituitary gonadotropins LH and FSH stimulate gonadal sex steroid hormone production directly by activating Leydig cells. Information is reviewed on the effects of progestin, androgens, and estrogens on global testicular gene expression patterns (microarray analysis), and on the molecular mechanisms by which steroids regulate specific candidate genes (identified by subtractive hybridization approaches) during early stages of testis maturation. Moreover, progestin and androgen effects on spermiation and milt hydration are discussed. Sex steroids mainly act via receptors expressed by Sertoli cells. One type of response is that Sertoli cells change growth factor expression, which subsequently modulates germ cell proliferation/differentiation via mechanisms yet to be characterised. Finally, we review data on germ cell autonomous processes, mainly derived from loss-of-function mutant fish lines, before identifying a number of focus areas for future research activities. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2009.02.013

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Publisher：Japan Science Support Foundation  

DOI： 10.5363/tits.15.6_87

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Other Link： https://jlc.jst.go.jp/DN/JALC/00356595960?from=CiNii

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press Inc.  

DOI： 10.1016/j.ygcen.2010.02.001

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Trypsin is well known as a pancreatic enzyme that is typically secreted into the intestine to digest proteins. We show in our current study, however, that trypsin is also a key factor in the control of spermatogenesis. A progestin in teleost fish, 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (DHP), is an essential component of the spermatogenesis pathway, particularly during the initiation of the first meiotic division. In the course of our investigations into the mechanisms underlying progestin-stimulated spermatogenesis, we identified that eel trypsinogen is upregulated in eel testis by DHP treatment. Trypsinogen is expressed in the Sertoli cells surrounding spermatogonia and in the membranes of spermatids and spermatozoa. Using an in vitro eel testicular culture system, we further analyzed the roles of trypsin in spermatogenesis. The inhibition of trypsin using specific antibodies or serine protease inhibitors was found to compromise DHP-induced spermatogenesis. A low dose of trypsin induces DNA synthesis and the expression of Spo11, a molecular marker of meiosis, in germ cells. By comparison, a higher dose of trypsin partially induced spermiogenesis. Furthermore, trypsin was detectable in the membranes of the spermatozoa and found to be associated with fertilization in fish. Our results thus demonstrate that trypsin and/or a trypsin-like protease is an essential and multifunctional factor in spermatogenesis.

DOI： 10.1073/pnas.0907631106

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOSCIENTIFICA LTD  

The precise mechanism and direct effects of arsenic on fish, particularly in reproduction, are not well clarified. The aim of this study is to investigate the direct influence of arsenic on fish spermatogenesis using the Japanese eel (Anguilla japonica) in vitro testicular organ culture system. Eel testicular fragments were cultured in vitro with 0.1-100 mu M arsenic with or without human chorionic gonadotropin (hCG) for 6 or 15 days at 20 degrees C. Arsenic treatment provoked a dose-dependent inhibition of hCG-induced germ cell proliferation as revealed by 5-bromo-2-deoxyuridine immunohistochemistry. Time-resolved fluorescent immunoassay showed that arsenic suppressed hCG-induced synthesis of 11-ketotestosterone (11-KT) in testicular fragments incubated with 0.0001-100 mu M arsenic and hCG for 18 h. A 0.1 mu M (7 mu g/l) dose of arsenic which is lower than the World Health Organization drinking water quality guideline of 10 mu g/l most effectively reduced 11-KT production. The hCG-induced synthesis of progesterone from pregnenolone was significantly inhibited by low doses of arsenic (0.1-1 mu M), implying an inhibition of 3 beta-hydroxysteroid dehydrogenase activity. In situ TUNEL assays indicated that germ cells undergo apoptosis at the highest dose of arsenic (100 mu M). An arsenic concentration-dependent increase in oxidative DNA damage was detected by 8-hydroxy-2'-deoxyguanosine (8-OHdG) immunohistochemistry. A peak in 8-OHdG index was observed in testicular fragments treated with 100 mu M arsenic and hCG consistent with the TUNEL results. These data suggest that low doses of arsenic may inhibit spermatogenesis via steroidogenesis suppression, while high doses of arsenic induce oxidative stress-mediated germ cell apoptosis. Reproduction (2009) 138 279-287

DOI： 10.1530/REP-09-0167

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Zinc (Zn) plays important roles in various biological activities but there is little available information regarding its functions in spermatogenesis. In our current study, we further examined the role of Zn during spermatogenesis in the Japanese eel ( Anguilla japonica). Human CG (hCG) was injected into the animals to induce spermatogenesis, after which the concentration of Zn in the testis increased in tandem with the progression of spermatogenesis. Staining of testicular cells with a Zn-specific fluorescent probe revealed that Zn accumulates in germ cells, particularly in the mitochondria of spermatogonia and spermatozoa. Using an in vitro testicular organ culture system for the Japanese eel, production of a Zn deficiency by chelation with N,N,N&apos;,N&apos;-tetrakis (2-pyridylemethyl) ethylenediamine ( TPEN) caused apoptosis of the germ cells. However, this cell death was rescued by the addition of Zn to the cultures. Furthermore, an induced deficiency of Zn by TPEN chelation was found to inhibit the germ cell proliferation induced by 11-ketotestosterone (KT), a fish specific androgen, 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP), the initiator of meiosis in fish, and estradiol-17 beta (E2), an inducer of spermatogonial stem-cell renewal. We also investigated the effects of Zn deficiency on sperm motility and observed that TPEN treatment of eel sperm suppressed the rate and duration of their motility but that co-treatment with Zn blocked the effects of TPEN. Our present results thus suggest that Zn is an essential trace element for the maintenance of germ cells, the progression spermatogenesis, and the regulation of sperm motility.

DOI： 10.1073/pnas.0900602106

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Cellulase activity has been detected in the digestive tract of earthworms. However, it has not been well clarified whether the origin of those cellulases are the earthworm themselves or the symbionts. In our study, zymogram analysis suggests that one cellulase (endo-beta-1,4-glucanase, EC3.2.1.4) mainly works to digest cellulose in Pheretima hilgendorfi. To identify the cellulase in P. hilgendorfi, we carried out cDNA cloning of the cellulase gene from the digestive tract. A novel cellulase gene was identified from the gut of earthworm. The cDNA encoding cellulase of P. hilgendorfi (phhEG) is 1606 bp with an open reading frame encoding a protein of 449 amino acid residues. The deduced amino acid sequence of P. hilgendorfi cellulase showed higher homology to invertebrate cellulases than bacterium cellulases belonging to the glycosyl hydrolase family (GHF) 9. The phhEG gene was detected in intestinal epithelium cell of mid-foregut using Northern blot and in situ hybridization. Similarly, specific cellulase activity against carboxymethyl cellulose (CMC) was significantly higher in midforegut tissue. Recombinant phhEG produced by wheat germ cell-free protein synthesis system had a cellulase activity which degrade CIVIC In zymogram analysis, the molecular size of cellulase was detected as a single band of 51 kDa from the whole gut contents extracts of P. hilgendorfi, and was very similar to the predicted molecular size of the mature phhEG protein. These results strongly suggested that the earthworm has the capacity to produce the endogenous and functional cellulase around the midforegut, and use this cellulase for their cellulose digestion with the support of intestinal caecum. (C) 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.soilbio.2009.01.016

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

Two gonadotropins (Gths), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), control gonadal steroidogenesis and gametogenesis in vertebrates, including teleost fish. Here, we report on the production of biologically active recombinant Fsh (rec-Fsh) and Lh (rec-Lh) in Japanese eel using Drosophila S2 cells. The three subunits composing Gths, i.e., glycoprotein hormone, alpha polypeptide (Cga), follicle-stimulating hormone, beta polypeptide (Fshb), and luteinizing hormone, beta polypeptide (Lhb), were at first independently produced and were proven to be glycosylated and secreted as the mature peptides. Each beta subunit, along with its Cga, was simultaneously coexpressed to produce heterodimeric rec-Fsh and rec-Lh that were subsequently highly purified. The biological activity of rec-Gths was demonstrated in various in vitro assays. The rec-Gths differentially activated their receptors, which resulted in an increase in 11-ketotestosterone (11KT) secretion, a differential alteration of gene expression of steroidogenic enzymes in immature testis, and the induction of the complete process of spermatogenesis in vitro. The data strongly suggest that Fsh and Lh differentially play important roles in the reproductive physiology of the Japanese eel. By contrast, these rec-Gths exhibited little activity in the gonad when administered in vivo. This difference between in vitro and in vivo bioactivity is probably due to the qualitative nature of glycosylation in S2 cells, which resulted in degradation of the recombinant protein in vivo. These differences in the carbohydrate moieties need to be elucidated and ameliorated.

DOI： 10.1095/biolreprod.108.070052

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

To estimate the influence of water contamination by arsenic (As) on reproduction of crustaceans in Vietnam, we collected wild freshwater crab Somanniathelphusa pax from the Mekong Delta area in Vietnam, investigated gonadal development, and measured As concentration in hepatopancreas. In female crab, vitellogenesis was delayed in association with the increase of As accumulation in hepatopancreas, whereas there was no significant correlation between testicular development and As accumulation in male crab. To clarify the effects of As on gonadal development of crustaceans, we investigated the effects of oral As administration on gonadal development in Japanese freshwater crab Geothelphusa dehaani. In male crab, the occurrence of spermatids and spermatozoa were predominantly observed in the control group, whereas the occurrence of spermatocytes increased after administration of 10 mu g/crab As for 3 months. On the other hand, in females, secondary yolk globule stages mainly occupied ovary of the control group. However, the primary yolk globule stage gradually increased after 10 mu g/crab As administration. Together these results indicate that it is possible that As contamination in water or food causes the delay of spermatogenesis and vitellogenesis in crustaceans.

DOI： 10.1007/s10646-008-0228-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC FRANCAISE D ICHTYOLOGIE  

Using cDNA subtraction and differential screening, we identified 25 genes that were differentially expressed by 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP) stimulation. Among these clones, eel trypsin and eel 11 beta-hydroxysteroid dehydrogenase short form (e11 beta-HSDsf) were analyzed. The results indicate that transcripts of eel trypsin and e11 beta-HSD in testis were induced by DHP stimulation. In vitro experiments revealed that trypsin induces initiation of meiosis and spermiogenesis. Moreover, cortisol enhanced the 11-ketotestesterone (11KT)-induced spermatogonial proliferation. From these results, we conclude that these two molecules are important for controlling spermatogenesis.

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Fertilization is an important process for the continuance of the species. In most of fish species, sperm fuses with an ovum through micropyle on the egg chorion because sperm does not have acrosome. In Japanese eel (Anguilla japonica), however, trypsin, a member of the serine protease family, is expressed in their testis by stimulation of 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP), which induces the initiation of meiosis, spermiation and sperm maturation in spermatogenesis. In this study, we investigated whether the trypsin-like protease exists in sperm in eel and kelp bass (Epinephelus bruneus Bloch) and it acts on fertilization in fish using sperm and eggs of both fishes. Tryptic activity was detected in the solubilized sperm membrane protein in both fishes. Using sperm of both fishes, treated with serine protease inhibitors or anti-eel trypsin antibody, artificial fertilization was performed. As a result, treatment with serine protease inhibitors or anti-eel trypsin antibody significantly decreased the fertilization rates in both fishes. These results suggest that trypsin-like protease exists in sperm membrane and plays an important role in fertilization of fish whose sperm does not have acrosome.

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The effects of Ph, Mo, As and Rb on spermatogenesis were investigated by using testicular organ culture of Japanese eel (Anguilla japonica). Treatment with these trace elements together with 11-ketotestosterone (KT) significantly inhibited KT-induced germ cells proliferation, suggesting that Mo, Rb, Ph and As directly interrupt KT-induced spermatogenesis. We also investigated the effects of As on in vitro KT synthesis in testis. KT production induced by human chorionic gonadotropin (hCG) was inhibited by low doses of As. This result suggested that As also may directly inhibit KT production.

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Using cDNA subtraction and differential screening, we identified 25 genes that were differentially expressed by 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP) stimulation. Among these clones, eel trypsin and eel 11 beta-hydroxysteroid dehydrogenase short form (e11 beta-HSDsf) were analyzed. The results indicate that transcripts of eel trypsin and e11 beta-HSD in testis were induced by DHP stimulation. In vitro experiments revealed that trypsin induces initiation of meiosis and spermiogenesis. Moreover, cortisol enhanced the 11-ketotestesterone (11KT)-induced spermatogonial proliferation. From these results, we conclude that these two molecules are important for controlling spermatogenesis.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC FRANCAISE D ICHTYOLOGIE  

Meiosis is an indispensable process of sexual reproduction. However, detailed information on the regulatory mechanisms that initiate meiosis is not available. Progestins are important steroids regulating final maturation in male and female vertebrates. In male teleosts, it is known that progestin induces spermiation and sperm maturation. However, a role for progestin in early spermatogenesis or meiosis has not yet been described. Using several in vitro gonadal culture systems in male and female, we have analyzed the role of 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP), a natural progestin in teleost fish, in controlling spermatogenesis and early oogenesis. DHP and its receptors were present in the testis at an early stage of spermatogenesis. Using eel testicular culture systems, DHP was shown to induce DNA replication in spermatogonia. Although 11-ketotestosterone, a known initiator of spermatogenesis, also stimulated DNA synthesis in spermatogonia, antibodies against DHP prevented DNA replication when added during the period in which meiosis was initiated. DHP treatment also induced the expression of meiosis specific markers, such as DmcI and Spo11. Furthermore, Spo11 expression and synaptonemal complexes (SC), specific features of the meiotic prophase, were detected in testicular fragments cultured with DHP in some germ cells that showed morphological characteristics of undifferentiated spermatogonia. In huchen and carp ovarian organ culture, DHP induced DNA synthesis of oogonia. In addition, the expression of Spo11 was observed histochemically and SC were ultra-microscopically detected in some germ cells. Therefore, DHP is also implicated in the regulation of early oogenesis from oogonial proliferation to initiation of the first meiotic division. These data suggest that DHP, a progestin, is an essential factor for the initiation of meiosis in both spermatogenesis and oogenesis.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ENDOCRINE SOC  

Mullerian inhibiting substance (MIS) is a glycoprotein belonging to the TGF-beta superfamily. In mammals, MIS is responsible for the regression of Mullerian ducts in the male fetus. However, the role of MIS in gonadal sex differentiation of teleost fish, which have no Mullerian ducts, has yet to be clarified. In the present study, we examined the expression pattern of mis and mis type 2 receptor (misr2) mRNAs and the function of MIS signaling in early gonadal differentiation in medaka (teleost, Oryzias latipes). In situ hybridization showed that both mis and misr2 mRNAs were expressed in the somatic cells surrounding the germ cells of both sexes during early sex differentiation. Loss-of-function of either MIS or MIS type II receptor (MISRII) in medaka resulted in suppression of germ cell proliferation during sex differentiation. These results were supported by cell proliferation assay using 5-bromo-2'-deoxyuridine labeling analysis. Treatment of tissue fragments containing germ cells with recombinant eel MIS significantly induced germ cell proliferation in both sexes compared with the untreated control. On the other hand, culture of tissue fragments from the MIS- or MISRII-defective embryos inhibited proliferation of germ cells in both sexes. Moreover, treatment with recombinant eel MIS in the MIS- defective embryos dose-dependently increased germ cell number in both sexes, whereas in the MISRII-defective embryos, it did not permit proliferation of germ cells. These results suggest that in medaka, MIS indirectly stimulates germ cell proliferation through MISRII, expressed in the somatic cells immediately after they reach the gonadal primordium.

DOI： 10.1210/en.2007-1535

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

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

DOI： 10.1095/biolreprod.107.062299

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Neobenedenia girellae, a monogenean skin parasite, shows low host specificity. N. girellae is an important pathogen in marine cultured fish such as yellowtail and amberjack. An effective control method is required but none has yet been established. To clarify the mechanisms of host specificity, we purified and identified the attachment-inducing substances of oncomiracidia from tiger puffer fish. The attachment-inducing substances were mainly included in skin mucous extract. Skin mucous extract lost its ability to induce attachment after boiling and/or exposure to the reducing agent dithiothreitol, suggesting that attachment-inducing substances are of a proteinaccous nature. Since lectins such as Con A, WGA, PHA-L, and PSA inhibited the induction of attachment, attachment-inducing proteins were suspected to be glycoproteins. Glycoproteins specifically interacting with Con A were collected and purified by anion exchange chromatography, resulting in two active peaks (peaks 3-A and 6). The active component in peak 3-A was identified as Wap 65-2 by N-terminal amino acid sequencing, while the glycoprotein in peak 6 could not be identified. These results suggested that oncomiracidia recognised Wap 65-2 and another glycoprotein of their host. (c) 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.ijpara.2007.04.024

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

Using two species of teleost fish, Japanese huchen (Hucho perryi) and common carp (Cyprinus carpio), we investigated whether sex steroids are involved in early oogenesis in vitro. Ovarian fragments were cultured to examine the effects of a progestin, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP), and an estrogen, estradiol-17 beta (E2). DHP and E2 significantly promoted DNA synthesis in ovarian germ cells, as judged by 5-bromo-2-deoxyuridine (BrdU) incorporation into these cells. Furthermore, to detect the initiation of the first meiotic division of early oogenesis, we assessed ultrastructurally the occurrence of synaptonemal complexes (SCs) and analyzed by immunohistochemistry the expression of a meiosis-specific marker, Spo11. In huchen, a higher percentage of oocytes with SC was seen in DHP-treated ovarian fragments than in control or E2-treated ovarian fragments. Spo11 was expressed in germ cells after DHP treatment of carp ovarian explants. These data suggest that the progression of germ cells through early oogenesis involves two sex steroids: E2, which acts directly on oogonial proliferation, and DHP, which acts directly on the initiation of the first meiotic division of oogenesis.

DOI： 10.1095/biolreprod.107.061408

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

To stimate the influence of water contaminants on fish reproduction in the Mekong Delta area, we sampled cultivated male catfish (Pangasianodon hypophthalmus), investigated testicular development, and measured persistent organic pollutants (POPs) and trace element levels in muscle and liver, respectively. Various testes sizes were observed although sampling took place during a short period. Histological analysis revealed that all developmental stages of germ cells were observed in catfish with large testis, whereas only necrotic spermatogonia but no other germ cells were observed in catfish with small testis. In small testis, furthermore, vacuolization and hypertrophy of Sertoli cells were observed. Measurement of POPs in muscle and trace elements in liver demonstrated that there were negative correlations between GSI and the concentrations of Pb, Mo, Rb and As. To clarify possible direct effects of Pb, Mo, Rb and As on spermatogenesis in fish, we investigated the effects of these trace elements on spermatogenesis using in vitro testicular organ culture of Japanese eel (Anguilla japonica). Treatment with each of the trace elements alone did not affect spermatogenesis. However, treatment with 10(-7) M of Pb, 10(-5) and 10(-4) M of Mo, 10(-5) -10(-3) M of Rb or 10(-5) M of As inhibited the spermatogenesis induced by 11-ketotestosterone (11 KT). Furthermore, treatment with 10(-4) M of As in combination with 11KT caused necrosis of testicular fragments. Taken together, these results are consistent with the hypothesis that Ph, Mo, Rb and As can exert inhibitory effects on spermatogenesis in catfish inhabiting the Mekong Delta area. (C) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.aquatox.2007.03.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Genes encoding spermatogenesis-related substance (eSRSs) show unique expression patterns during spermatogenesis. To analyze their function, we developed a new assay system using gene transfer techniques combined with coculture of the eel germ-somatic cells. First, we investigated the efficacy of in vitro electroporation transfer of gene into germ-somatic cell pellets using green fluorescent protein (GFP) gene. Second, in order to define the function of the eSRSs, we electrophoretically transferred eel spermatogonial stem cell renewal factor (eSRS34) and eel spermatogenesis-preventing substance (eSRS21) genes into germ-somatic cell pellets. Presence of the transferred cDNA was examined by reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, proliferating cells were detected histologically, after labeling with BrdU. Transfer of the eSRS34 gene induced spermatogonial stem cell renewal in the pellets. Moreover, 11-ketotestosterone (11-KT) treatment stimulated the proliferation of spermatogonia, which resulted in the appearance of late type B spermatogonia in the pellets. The proliferation of spermatogonia by 11-KT stimulation was suppressed by transfer of the eSRS21 gene. These results indicate that the transferred eSRS34 and 21genes were functional in the pellets. Thus, an efficient in vitro gene transfer technique for coculture system of germ and somatic cell of Japanese eel was established.

DOI： 10.1002/mrd.20653

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

Neobenedenia girellae, a monogenean, is an important pathogen in marine cultured fish such as yellowtail and amberjack. An effective control method is required but none has yet been established. Aiming to establish a new control method by interfering with the gametogenesis of N. girellae, we focused on vasa (vas)-related genes that are expressed exclusively in the germline granules in Drosophila, Caenorhabditis elegans and other animals. Three vas-related genes (N. girellae vasa-like gene, Ngvlg1, Ngvlg2 and Ngvlg3) were isolated by PCR and Ngvlg1 and Ngvlg2 were shown to be expressed only in germ cells. We demonstrated that introduction of double-stranded Ngvlg1 or Ngvlg2 RNA by soaking resulted in partial or complete loss of germ cells. Moreover, the hatching rate of eggs from animals showing partial loss of germ cells decreased significantly. These results suggest that Ngvlg1 and Ngvlg2 are essential genes for germ cell. quantity and quality. The possibility that a new control method can be developed by controlling gametogenesis of N. girellae was proven, because sterilised N. girellae could be produced. (c) 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.ijpara.2006.11.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CAMBRIDGE UNIV PRESS  

This study investigated a new effective method for controlling the capsalid monogenean Neobenedenia girellae. We examined in vitro and in vivo the effect on the percentage survival of N. girellae in buffers containing different metallic ions. Decreased survival was observed in buffer solutions lacking two ions. In particular, the percentage survival of N. girellae was significantly decreased after 10 min exposure to buffer containing neither Ca(2+) nor Mg(2+). Transmission electron microscopic observations showed that treatment with this buffer disrupted intercellular junctions. This significant effect on percentage survival of N. girellae using Ca(2+)/Mg(2+) -free buffer was confirmed in an in vivo assay. Ca(2+)/Mg(2+)-free buffer had no effect on the condition of the host, spotted halibut Verasper variegates (Pleuronectidae). These results suggest that treatment with Ca(2+)/Mg(2+) -free buffer is a new effective control method, which could replace existing control methods.

DOI： 10.1017/S0031182006001430

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

In mammals, a multitude of studies have shown that anti-Mullerian hormone (AMH/AMH), apart from inducing Mullerian duct regression during male sexual differentiation, exerts inhibitory effects on male and female gonadal steroidogenesis and differentiation. However, in lower vertebrates like teleost fish, the function of AMH/AMH has been far less explored. As a first step to unravel its potential role in reproduction in teleost fish, we isolated and characterised the AMH gene in the European sea bass (sb), Dicentrachus labrax, determined putative regulatory elements of its 5&apos;-flanking region, and analysed its gene expression and those of altematively-spliced transcripts. The characterisation of sb-AMH revealed distinct features that distinguishes it from mammalian and bird AMH, suggesting a high rate of diversification of AMH during vertebrate evolution. It contained 7 exons that were divided by 6 introns, of which the last intron (intron vi) was localised only a few nucleotides upstream of the putative peptide cleavage site. The guanine and cytosine content of the open reading frame (ORF) was 52.7% and thus notably lower than that of bird and mammalian AMH. Sb-AMH cDNA was 2045 base pairs (bp) long, containing an ORF of 1599 bp encoding 533 amino acids. Deduced amino acid similarities of the conserved, carboxyterminal domain were highest with AMH in Japanese flounder (84.2%) and lowest with chicken AMH (45.5%). In the proximal promoter sequence of sb-AMH, a steroidogenic factor-1 (SF-1) binding site was present; however other regulatory sequences essential for transcriptional activation of AMH in mammals were absent. Likewise, there was no sequence homology to an SF3A2 sequence within the first 3200 bp upstream of the sb-AMH translation start site. Gene expression of sb-AMH and of alternatively-spliced sb-AMH transcripts were analysed in male and female juvenile and adult gonads as well as in somatic tissues of juvenile males. sb-AMH expression was highest in juvenile testis, but still remarkably high in juvenile ovaries and adult testis, as well as in brain, pituitary, and heart of juvenile male sea bass. Apart from adult ovary, levels of alternatively-spliced sb-AMHexonII/-99 were marginal in comparison with sb-AMH. In contrast, the transcript variant sb-AMHexonVII/+5 was expressed to a similar extent as sb-AMH in all tissues examined. The results of this work have provided the basis for future studies concerning the regulation and function of AMH/AMH in this species. (c) 2006 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.gene.2006.10.018

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ENDOCRINE SOC  

In fish spermatogenesis, the main action of progestins is generally regarded as the induction of sperm maturation. Our previous in vitro study demonstrated that a progestin, 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP), induced the initiation of meiosis in spermatogenesis in the Japanese eel (Anguilla japonica). In the present study, to elucidate the molecular mechanisms underlying the action of DHP, we attempted to clone cDNAs encoding genes whose expression was induced by DHP in eel testis, using cDNA subtraction. One of the cDNAs we isolated encodes eel 11 beta-hydroxysteroid dehydrogenase short form (e11 beta-HSDsf), and Northern blot and RT-PCR analysis showed that transcripts of e11 beta-HSDsf in testis were induced by DHP. The recombinant e11 beta-HSDsf had 11 beta-dehydrogenase activity, metabolizing cortisol to cortisone, and 11 beta-hydroxytestosterone to 11-ketotestosterone (11-KT). In vitro experiments revealed that eel immature testis had 11 beta-dehydrogenase activity, and DHP treatment enhanced the activity. To understand the role of 11 beta-HSD in spermatogenesis, we examined the direct effects of cortisol on eel spermatogenesis using an organ culture system. Cortisol induced DNA replication in spermatogonia and enhanced the spermatogonial proliferation induced by 11-KT. However, excess cortisol inhibited proliferation. In addition, 11-KT production was induced in testicular fragments incubated with cortisol. These results suggest that optimal levels of cortisol induced spermatogonial mitosis by increasing 11-KT production. Furthermore, two possible roles of DHP on spermatogenesis, via the up-regulation of 11 beta-HSD expression, are suggested: positive feedback control of 11-KT production and the modulation of cortisol levels to protect testes from excess circulating cortisol.

DOI： 10.1210/en.2006-0391

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Meiosis is an indispensable process of sexual reproduction. However, detailed information on the regulatory mechanisms that initiate meiosis is not available. Progestins are important steroids regulating final maturation in male and female vertebrates. In male teleosts, it is known that progestin induces spermiation and sperm maturation. However, a role for progestin in early spermatogenesis or meiosis has not yet been described. In this study, we examined the functions of progestin on the initiation of meiosis in male Japanese eel. A natural progestin in teleost fish 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (DHP) and its receptors were present in the testis at an early stage of spermatogenesis. By using an eel testicular culture system, DHP was shown to induce DNA replication in spermatogonia. Although 11-ketotestosterone, a known initiator of spermatogenesis, also stimulated DNA synthesis in spermatogonia, antibodies against DHP prevented DNA replication when added during the period in which meiosis was initiated. DHP treatment also induced the expression of meiosis-specific markers, such as Dmcl and Spo11. Furthermore, Spo11 expression and synaptonemal complexes, specific features of the meiotic prophase, were detected in testicular fragments cultured with DHP in some germ cells that showed morphological characteristics of undifferentiated spermatogonia. We conclude that DHP, a progestin, is an essential factor for the initiation of meiosis.

DOI： 10.1073/pnas.0508419103

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

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 Spo11. 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. (C) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cbpb.2005.12.008

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Endocrine disrupters have been recognized to interfere with endocrine systems that regulate reproduction, for example, by mimicking or inhibiting the action of endogenous sex steroid hormones including estradiol-17beta (E2). In the present study, we examined the effect of an endocrine disrupter, para-nonylphenol (p-NP) on spermatogenesis, and compared it with the action of E2, using an eel testicular organ culture system. p-NP alone stimulated early spermatogonial renewal in the same manner as E2. Neither induced further progress in spermatogenesis. In the presence of 11-ketotestosterone (11-KT), the major androgen in teleosts, p-NP did not prevent the 11-KT-induced progress in spermatogenesis. However, this treatment enlarged the Sertoli cells. Electron microscopic observation revealed that enlarged Sertoli cells contained well-developed organelles. Moreover, the proportion of germ cells appeared to have decreased as a result of Sertoli cell hypertrophy. These results clearly show that p-NP has an effect on Sertoli cells in the presence of an androgen (11-KT), potentially disturbing 11-KT-induced spermatogenesis. (C) 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.aquatox.2004.10.015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ENDOCRINE SOC  

Spermatogonial mitosis can be subdivided into two processes: spermatogonial stem cell renewal and spermatogonial proliferation toward meiosis. Recently it has been indicated that estrogen, estradiol-17beta, is involved in regulating the renewal of spermatogonial stem cells in eel. To determine the genes that directly regulate this process, we used expression screening to identify genes whose expression is regulated by estradiol-17beta in testes. We detected a previously unidentified cDNA clone that is up-regulated by estradiol-17beta stimulation and named it eel spermatogenesis-related substances 34 (eSRS34) cDNA. Homology searching showed that eSRS34 shares amino acid sequence similarity with human platelet-derived endothelial cell growth factor. We examined the function of eSRS34 using several in vitro systems. Recombinant eSRS34 produced by a baculovirus system induced spermatogonial mitosis in testicular organ culture. Furthermore, the addition of an antibody specific for eSRS34 prevented spermatogonial mitosis induced by estradiol-17beta stimulation in a germ cell/somatic cell coculture system. We therefore conclude that eSRS34 is a "spermatogonial stem cell renewal factor."

DOI： 10.1210/en.2003-0800

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER-VERLAG  

Differential display of mRNA was used to identify an upregulated gene in ovaries of artificially maturing Japanese eels (Anguilla japonica). Accordingly, mitochondrial (mt) cytochrome b, whose transcript levels increased from early to late vitellogenesis, was isolated, cloned and sequenced. Temporal trends in artificially maturing eels were compared with those in naturally and artificially maturing New Zealand eels (longfinned eel, Anguilla dieffenbachii; shortfinned eel, Anguilla australis) by Northern blot and in situ hybridization analysis to rule out any experimental artifacts. An increase in ovarian mt cytochrome b signals was seen when comparing immature and midvitellogenic longfinned eels, but not immature and early vitellogenic shortfinned eels, from the wild. Long-term captivity yielded reduced target mRNA levels, but abundance increased after hormonal induction of vitellogenesis. These results imply that the increase in mt cytochrome b mRNA levels during artificial maturation reflects natural development, although its onset appears to be brought forward during artificial maturation in the Japanese eel. It is suggested that increased mt cytochrome b mRNA levels result from both mitochondrial replication and increased transcription, and that they reflect the build-up of machinery for enhanced ATP-synthesis at some stage of oogenesis and/or early zygote development.

DOI： 10.1007/s00360-002-0304-x

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER-VERLAG TOKYO  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KLUWER ACADEMIC PUBL  

The present study was carried out to investigate whether several sex steroid hormones are able to induce oogonial proliferation or initiation of meiotic division I in vitro. The results showed that 17beta-estradiol (E2) and 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17alpha, 20beta-DHP) were able to induce the DNA synthesis of ovarian germ cells. The percentage of oocytes in 17alpha, 20beta-DHP treated fragments was significantly higher than that in the other treatments. From these results, we conclude that endogenous E2 and 17alpha, 20beta-DHP synthesized in ovary play significant roles in the proliferation of oogonia and the initiation of meiotic division I in early oogenesis, respectively.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KLUWER ACADEMIC PUBL  

Fish spermatogenesis, from spermatogonial stem-cell renewal to sperm maturation, is controlled by the sex steroid hormones. Mitotic divisions of spermatogonia can be categorized by spermatogonial stem cell renewal and spermatogonial proliferation. Spermatogonial renewal is regulated by estradiol-17beta (E-2; the natural estrogen in vertebrates), and spermatogonial proliferation toward meiosis is promoted by 11-ketotestosterone (11-KT), the main androgen in teleost. The action of E2 and 11-KT is mediated by other factors produced by Sertoli cells; E2 is mediated by spermatogonial stem-cell renewal factor and 11-KT is mediated by spermatogenesis preventing substance and activin B. Although 11-KT also inducemeiosis and spermiogenesis, the control mechanisms of these processe are not clear. After spermiogenesis, immature spermatozoa undergo sperm maturation. Sperm maturation is regulated by 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP), which is progestin in teleost. The DHP acts directly on spermatozoa to active the carbonic anhydrase existed in the spermatozoa. This enzymatic activation causes an increase in the seminal plasma pH, enabling spermatozoa to motile.

DOI： 10.1023/B:FISH.0000030522.71779.47

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

Under fresh-water cultivation conditions, spermatogenesis in the Japanese eel is arrested at an immature stage before initiation of spermatogonial proliferation. A single injection of human chorionic gonadotropin can, however, induce complete spermatogenesis, which suggests that spermatogenesis-preventing substances may be present in eel testis. To determine whether such substances exist, we have applied a subtractive hybridisation method to identify genes whose expression is suppressed after human chorionic gonadotropin treatment in vivo. We found one previously unidentified cDNA clone that was downregulated by human chorionic gonadotropin, and named it 'eel spermatogenesis related substances 21' (eSRS21). A homology search showed that eSRS21 shares amino acid sequence similarity with mammalian and chicken Mullerian-inhibiting substance. eSRS21 was expressed in Sertoli cells of immature testes, but disappeared after human chorionic gonadotropin injection. Expression of eSRS21 mRNA was also suppressed in vitro by 11-ketotestosterone, a spermatogenesis-inducing steroid in eel. To examine the function of eSRS21 in spermatogenesis, recombinant eSRS21 produced by a CHO cell expression system was added to a testicular organ culture system. Spermtogonial proliferation induced by 11 ketotestosterone in Nitro was suppressed by recombinant eSRS21. Furthermore, addition of a specific anti-eSRS21 antibody induced spermatogonial proliferation in a germ cell/somatic cell co-culture system. We conclude that eSRS21 prevents the initiation of spermatogenesis and, therefore, suppression of eSRS21 expression is necessary to initiate spermatogenesis. In other words, eSRS21 is a spermatogenesis-preventing substance.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

There is much information on oogenesis from the resumption of the first meiotic division to oocyte maturation in many vertebrates; however, there have been very few studies on early oogenesis from oogonial proliferation to the initiation of meiosis. In the present study, we investigated the histological changes during early oogenesis in barfin flounder (Verasper moseri). In fish with a total length (TL) of 50mm (TL 50mm fish), active oogonial proliferation was observed. In TL 60mm fish, oocytes with synaptonemal complexes were observed. Before the initiation of active oogonial proliferation, somatic cells which surrounded a few oogonial germ cells, started to proliferate to form the oogonial cysts that accompanied oogonial proliferation. In TL 70mm fish, however, the cyst structure of the oocyte was gradually broken by the invagination of somatic cells, and finally the oocyte became a single cell surrounded by follicle cells. Upon comparison of nuclear size, DNA-synthesizing germ cells could be divided into two types: small nuclear cells and large nuclear cells. Based on histological observation, we propose that the small nuclear cells were in the mitotic prophase of oogonia and the large nuclear cells were in the meiotic prophase of oocytes, and that the nuclear size increases upon the initiation of meiosis.

DOI： 10.2108/zsj.19.557

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DOI： 10.2331/fishsci.68.sup2_1269

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER TOKYO  

The present study examined the ability of various steroids and hormones to induce in vitro spermatogonial proliferation in cultured testicular fragments of two teleost species. Results indicate that the three steroids 11-ketotestosterone (11-KT), 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-DHP) and 17 beta-estradiol (E-2) and salmon pituitary glycoprotein fraction (SPG) were able to induce DNA synthesis, spermatogonial renewal and/or spermatogonial proliferation in Japanese huchen (Hucho perryi), and 100 pg/mL of 17 alpha,20 beta-DHP was sufficient to induce spermatogonial proliferation in the Japanese eel (Anguilla japonica). From these results, we concluded that 11-KT is necessary for the initiation of spemtogenesis; estrogen is necessary for spermatogonial stem cell renewal; and 17 alpha,20 beta-DHP promoted DNA synthesis, and plays a significant role in the proliferation of spermatogonia during mitosis.

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Language：English   Publisher：KLUWER ACADEMIC PUBL  

Three major phases compose spermatogenesis: mitotic proliferation of spermatogonia, meiosis of spermatocytes, and spermiogenesis, the restructuring of spermatids into flagellated spermatozoa. The process is fuelled by stem cells that, when dividing, either self-renew or produce spermatogonia that are committed to proliferation, meiosis, and spermiogenesis. During all phases, germ cells are in close contact with and require the structural and functional support of Sertoli cells. In contrast to germ cells, these somatic cells express receptors for sex steroids and follicle-stimulating hormone (FSH), the most important hormones that regulate spermatogenesis. A typical Sertoli cell response to an endocrine stimulus would be to change the release of a growth factor that would then mediate the hormone's effect to the germ cells. Recent studies in the Japanese eel have shown, for example, that in the absence of gonadotropin Sertoli cells produce a growth factor (an orthologue of anti-Mullerian hormone) that restricts stem cell divisions to the self-renewal pathway; also estrogens stimulate stem cell renewal divisions but not spermatogonial proliferation. Gonadotropin or 11-ketotestosterone (11-KT) stimulation, however, induces spermatogonial proliferation, which is in part mimicked by another Sertoli cell-derived growth factor (activin B). Since FSH (besides luteinizing hormone, LH) stimulates steroidogenesis in fish, and since FSH is the only gonadotropin detected in the plasma of sexually immature salmonids, increased FSH signalling may be sufficient to initiate spermatogenesis by activating both Sertoli cell functions and 11-KT production. Another important androgen is testosterone (T), which seems to act via feedback mechanisms that can compromise FSH-dependent signalling or steroidogenesis. The testicular production of T and 11-KT therefore needs to be balanced adequately. Further research is required to elucidate in what way(s) 11-KT stimulates later stages of development, such as entry into meiosis and spermiogenesis. At this period, LH becomes increasingly important for the regulation of androgen production. Results from mammalian models suggest that during the later phases, the control of germ cell apoptosis via Sertoli cell factors is an important regulatory mechanism. In many species, sperm cells cannot fertilize eggs until having passed a maturation process known as capacitation, which includes the acquisition of motility. Progestins that are produced under the influence of LH appear to play an important role in this context, which involves the control of the composition of the seminal plasma (e.g., pH values).

DOI： 10.1023/A:1023303427191

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

DOI： 10.2108/zsj.19.87

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In order to check the quality of in vitro spermatogenesis of Japanese eel, in vitro 11-ketotestosterone (11-KT) induced spermatogenesis was compared with in vivo spermatogenesis induced by a single injection of human chorionic gonadotropin (hCG) in detail. DNA contents of germ cells from in vitro and in vivo testicular fragments were compared using flow cytometry. Since the in vitro result of flow cytometry showed prominent 1C peak including spermatozoa and spermatids, the reduction of DNA by meiosis was assumed to progress normally, (i.e., haploid spermatozoa were produced in this in vitro system). In the testes of in vitro culture, however, spermatozoa were not released into lumen. Furthermore, the number of mitotic divisions of the in vitro experiment (6 divisions) was fewer than that of in vivo (10 divisions). In electron microscopy observations, both of in vivo and in vitro spermatozoon had a crescent-shaped nucleus with a flagellum, and a single large spherical mitochondrion. However, the elongation of the sperm head was not sufficient and the mitochondrion was not always located at the anterior end as is observed for the spermatozoa obtained from hCG injected eels. Eel spermatogenesis related substance-11 (eSRS11) is homologue of histone H1 which is up-regulated during spermatogenesis. Using this probe, in vitro spermatogenesis was also evaluated in molecular levels. In Northern blot analysis, eSRS11 mRNA was detected in both in vivo and in vitro testes. However, the expression of in vitro was much weaker than that of in vivo. These differences indicate that the stimulation of 11-KT is not sufficient, and another factors are needed to induce complete spermatogenesis in vitro.

DOI： 10.2108/zsj.19.321

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER TOKYO  

The present study examined the ability of various steroids and hormones to induce in vitro spermatogonial proliferation in cultured testicular fragments of two teleost species. Results indicate that the three steroids 11-ketotestosterone (11-KT), 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-DHP) and 17 beta-estradiol (E-2) and salmon pituitary glycoprotein fraction (SPG) were able to induce DNA synthesis, spermatogonial renewal and/or spermatogonial proliferation in Japanese huchen (Hucho perryi), and 100 pg/mL of 17 alpha,20 beta-DHP was sufficient to induce spermatogonial proliferation in the Japanese eel (Anguilla japonica). From these results, we concluded that 11-KT is necessary for the initiation of spemtogenesis; estrogen is necessary for spermatogonial stem cell renewal; and 17 alpha,20 beta-DHP promoted DNA synthesis, and plays a significant role in the proliferation of spermatogonia during mitosis.

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Under fresh-water cultivation conditions, spermatogenesis in the Japanese eel is arrested at an immature stage before initiation of spermatogonial proliferation. A single injection of human chorionic gonadotropin can, however, induce complete spermatogenesis, which suggests that spermatogenesis-preventing substances may be present in eel testis. To determine whether such substances exist, we have applied a subtractive hybridisation method to identify genes whose expression is suppressed after human chorionic gonadotropin treatment in vivo. We found one previously unidentified cDNA clone that was downregulated by human chorionic gonadotropin, and named it 'eel spermatogenesis related substances 21' (eSRS21). A homology search showed that eSRS21 shares amino acid sequence similarity with mammalian and chicken Müllerian-inhibiting substance. eSRS21 was expressed in Sertoli cells of immature testes, but disappeared after human chorionic gonadotropin injection. Expression of eSRS21 mRNA was also suppressed in vitro by 11-ketotestosterone, a spermatogenesis-inducing steroid in eel. To examine the function of eSRS21 in spermatogenesis, recombinant eSRS21 produced by a CHO cell expression system was added to a testicular organ culture system. Spermtogonial proliferation induced by 11-ketotestosterone in vitro was suppressed by recombinant eSRS21. Furthermore, addition of a specific anti-eSRS21 antibody induced spermatogonial proliferation in a germ cell/somatic cell co-culture system. We conclude that eSRS21 prevents the initiation of spermatogenesis and, therefore, suppression of eSRS21 expression is necessary to initiate spermatogenesis. In other words, eSRS21 is a spermatogenesis-preventing substance.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER TOKYO  

The present study examined the ability of various steroids and hormones to induce in vitro spermatogonial proliferation in cultured testicular fragments of two teleost species. Results indicate that the three steroids 11-ketotestosterone (11-KT), 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-DHP) and 17 beta-estradiol (E-2) and salmon pituitary glycoprotein fraction (SPG) were able to induce DNA synthesis, spermatogonial renewal and/or spermatogonial proliferation in Japanese huchen (Hucho perryi), and 100 pg/mL of 17 alpha,20 beta-DHP was sufficient to induce spermatogonial proliferation in the Japanese eel (Anguilla japonica). From these results, we concluded that 11-KT is necessary for the initiation of spemtogenesis; estrogen is necessary for spermatogonial stem cell renewal; and 17 alpha,20 beta-DHP promoted DNA synthesis, and plays a significant role in the proliferation of spermatogonia during mitosis.

DOI： 10.2331/fishsci.68.sup1_681

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DOI： 10.2331/fishsci.68.sup2_1269

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Language：English   Publisher：ZOOLOGICAL SOC JAPAN  

The Japanese eel has two characteristics advantageous for the study of the mechanisms controlling spermatogenesis. One is the possibility of artificial induction of the complete process of spermatogenesis from spermatogonial proliferation to spermiogenesis by exogenous gonadotropin injection, and the other is the possibility of inducing this process in an in vitro testicular organ culture or germ-Sertoll cell coculture system. Using the eel system, we analyzed the control mechanisms of spermatogenesis. In Japanese eel, the whole process of spermatogenesis is regulated by several sex steroid hormones. Spermatogonial stem cell renewal is promoted by estradiol-17 beta (the natural estrogen in vertebrates). Spermatogonial proliferation can be induced by 11-ketotestosterone, the main androgen in teleost. IGF-I is necessary for the action of 11-ketotestosterone in the initiation of spermatogenesis. The action of 11-ketotestosterone is mediated by other factors, such as activin B, produced by Sertoli cells. Although 11-ketotestosterone also induce meiosis and spermiogenesis, the control mechanisms of these processes are not clear. After spermiogenesis, immature spermatozoa undergo sperm maturation, thereby becoming capable of fertilization. Sperm maturation is regulated by 17 alpha ,20 beta -dihydroxy-4-pregnen-3-one (17 alpha ,20 beta -DP), which is progestogen in teleosts. The 17 alpha ,20 beta -DP acts directly on spermatozoa to activate the carbonic anhydrase existed in the spermatozoa. This enzymatic activation causes an increase in the seminal plasma pH, enabling spermatozoa to motile.

DOI： 10.2108/zsj.18.1055

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

In higher vertebrates, considerable progress has been made in understanding the endocrine regulation of puberty; however, in teleosts, the regulatory mechanisms of spermatogenesis during the first annual cycle remain unclear. The present study was conducted to understand the regulatory mechanisms of spermatogenesis throughout the different stages of the first spermatogenic cycle and to check the ability of various steroids and hormones to induce in vitro spermatogonial proliferation in Japanese huchen (Hucho perryi). The results indicate that the serum level of 11-ketotestosterone (11-KT) was positively associated with germ cell type; the level first began to rise with the appearance of late-type B spermatogonia and continued to increase gradually throughout the active spermatogenic stages and spermiogenesis, reaching a peak value 2 wk before spawning, and then declined. During the spermatogenic stages, the serum concentration of 17 alpha ,20 beta -dihydroxy-4-pregnen-3-one (17 alpha ,20 beta -DP) was undetectable. Only a small peak was detected with the appearance of spermatocytes and spermatids, and at the time of spawning, the level increased dramatically, reaching its maximum value with the onset of milt production. Despite the high variation in serum levels of 17 beta -estradiol (E-2) both between months and among the individuals, E-2 was found during the whole reproductive cycle. From these results, we concluded that 1) 11-KT is necessary for the initiation of spermatogenesis and sperm production, and it probably plays a role in spermiation, 2) 17 alpha ,20 beta -DP is essential for the final maturation stage, could play a significant role in the mitosis phase and meiosis process, and probably participates in the regulation of spawning behavior, and 3) estrogen is an indispensable male hormone that plays a physiological role in some aspects of testicular functions, especially during the mitotic phase. The three steroids were also able to induce DNA synthesis, spermatogonial renewal, and/or spermatogonial proliferation in vitro.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

In higher vertebrates, considerable progress has been made in understanding the endocrine regulation of puberty; however, in teleosts, the regulatory mechanisms of spermatogenesis during the first annual cycle remain unclear. The present study was conducted to understand the regulatory mechanisms of spermatogenesis throughout the different stages of the first spermatogenic cycle and to check the ability of various steroids and hormones to induce in vitro spermatogonial proliferation in Japanese huchen (Hucho perryi). The results indicate that the serum level of 11-ketotestosterone (11-KT) was positively associated with germ cell type; the level first began to rise with the appearance of late-type B spermatogonia and continued to increase gradually throughout the active spermatogenic stages and spermiogenesis, reaching a peak value 2 wk before spawning, and then declined. During the spermatogenic stages, the serum concentration of 17 alpha ,20 beta -dihydroxy-4-pregnen-3-one (17 alpha ,20 beta -DP) was undetectable. Only a small peak was detected with the appearance of spermatocytes and spermatids, and at the time of spawning, the level increased dramatically, reaching its maximum value with the onset of milt production. Despite the high variation in serum levels of 17 beta -estradiol (E-2) both between months and among the individuals, E-2 was found during the whole reproductive cycle. From these results, we concluded that 1) 11-KT is necessary for the initiation of spermatogenesis and sperm production, and it probably plays a role in spermiation, 2) 17 alpha ,20 beta -DP is essential for the final maturation stage, could play a significant role in the mitosis phase and meiosis process, and probably participates in the regulation of spawning behavior, and 3) estrogen is an indispensable male hormone that plays a physiological role in some aspects of testicular functions, especially during the mitotic phase. The three steroids were also able to induce DNA synthesis, spermatogonial renewal, and/or spermatogonial proliferation in vitro.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Society for the Study of Reproduction  

In higher vertebrates, considerable progress has been made in understanding the endocrine regulation of puberty
however, in teleosts, the regulatory mechanisms of spermatogenesis during the first annual cycle remain unclear. The present study was conducted to understand the regulatory mechanisms of spermatogenesis throughout the different stages of the first spermatogenic cycle and to check the ability of various steroids and hormones to induce in vitro spermatogonial proliferation in Japanese huchen (Hucho perryi). The results indicate that the serum level of 11-ketotestosterone (11-KT) was positively associated with germ cell type
the level first began to rise with the appearance of late-type B spermatogonia and continued to increase gradually throughout the active spermatogenic stages and spermiogenesis, reaching a peak value 2 wk before spawning, and then declined. During the spermatogenic stages, the serum concentration of 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP) was undetectable. Only a small peak was detected with the appearance of spermatocytes and spermatids, and at the time of spawning, the level increased dramatically, reaching its maximum value with the onset of milt production. Despite the high variation in serum levels of 17β-estradiol (E2 both between months and among the individuals, E2 was found during the whole reproductive cycle. From these results, we concluded that 1) 11-KT is necessary for the initiation of spermatogenesis and sperm production, and it probably plays a role in spermiation, 2) 17α,20β-DP is essential for the final maturation stage, could play a significant role in the mitosis phase and meiosis process, and probably participates in the regulation of spawning behavior, and 3) estrogen is an indispensable male hormone that plays a physiological role in some aspects of testicular functions, especially during the mitotic phase. The three steroids were also able to induce DNA synthesis, spermatogonial renewal, and/or spermatogonial proliferation in vitro.

DOI： 10.1095/biolreprod65.4.1057

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Zoological Society of Japan  

In male Japanese eels, eel spermatogenesis-related substance (eSRS) 3 and 4, having high sequence-similarities to zona pellucida protein (ZP) 2 and ZP3, respectively, are down-regulated by gonadotropin stimulation, with their transcripts disappearing upon the initiation of spermatogenesis. Using Northern blot analysis, we investigated the expression of eSRS3 and 4 mRNA in the developing ovary and the liver of SPH (salmon pituitary homogenate)-injected female eels. Both transcripts were detected in the ovary, but not in the liver. When the eel ovary was subjected to in situ hybridization using eSRSS and 4 cRNA probes, the cytoplasm of previtellogenic oocytes showed a strong signal in comparison with the weak signal in vitellogenic oocytes. Furthermore, stronger signals were observed in the chromatin-nucleolus and the perinucleolus stages than in the oil-droplet stage. Subsequently, we synthesized peptides that were deduced from eSRSS and 4 cDNAs and generated specific antibodies against them. Staining of the cytoplasm of oocytes in the previtellogenic stage and of egg envelopes in the vitellogenic stage occurred when these antibodies were used in an immunohistochemical analysis. These expression patterns in the ovary suggest that eSRSS and 4 are components of the eel egg envelope.

DOI： 10.2108/zsj.17.1297

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Blackwell Publishing  

To assess the number of mitotic divisions in fish spermatogonia, an easy method was devised which can estimate the number of mitotic divisions by only counting the cells in the cross-section of a cyst at its largest diameter. Using clay to simulate spermatogonial cysts, we obtained results which related the number of cells in a cross-section of a cyst at its largest diameter to the total number of cells in the same cyst. The results were then compared to the number of mitotic divisions observed in seven species. The number of divisions estimated from the simulation experiment coincided exactly with the observed values in all species with spherical shaped cysts. However, the counts determined in fish with elliptical shaped cysts differed from the expected values. As a result, it is clear that the obtained values from the simulation experiment are valid for the estimation of the number of mitotic divisions.

DOI： 10.1046/j.1444-2906.2000.00047.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

In this study, we examined the in vitro effects of insulin-like growth factor I (IGF-I) in the presence or absence of 11-keto-testosterone (11-KT: the spermatogenesis-inducing hormone) on the proliferation of Japanese eel (Anguilla japonica) testicular germ cells. Initially, a short-term culture (15 days) of testicular tissue with only type A and early type B spermatogonia (preproliferated spermatogonia) was carried out in Leibovitz-15 growth medium supplemented with different concentrations of recombinant human IGF (rhIGF)-I or -II in the presence or absence of 10 ng/ml of 11-KT. Late type B spermatogonia (proliferated spermatogonia) were observed in treatments of 100 ng/ml of both rhIGF-I and -II in combination with 11-KT, indicating the onset and progression of spermatogenesis. In all tested rhIGF-I concentrations (except 0.1 ng/ml) supplemented with 11-KT, late type 8 spermatogonia were detected in at least one individual. Then, we proceeded with an in vitro 45-day culture of testicular tissue with 100 ng/ml of rhIGF-I in the presence or absence of 10 ng/ml of 11-KT to test the long-term effects of rhIGF-I on the spermatogenetic cycle. The presence of all types of germ cells, including spermatozoa, in the testis cultured with the admixture of the two hormones indicated that the germ cells underwent complete spermatogenesis whereas no germ cell proliferation was observed when the rhIGF-I was applied alone. These results suggest that IGF-I in the presence of 11-KT plays an essential role in the onset, progress, and regulation of spermatogenesis in the testis of the Japanese eel.

DOI： 10.1095/biolreprod61.4.944

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

In this study, we examined the in vitro effects of insulin-like growth factor I (IGF-I) in the presence or absence of 11-keto-testosterone (11-KT: the spermatogenesis-inducing hormone) on the proliferation of Japanese eel (Anguilla japonica) testicular germ cells. Initially, a short-term culture (15 days) of testicular tissue with only type A and early type B spermatogonia (preproliferated spermatogonia) was carried out in Leibovitz-15 growth medium supplemented with different concentrations of recombinant human IGF (rhIGF)-I or -II in the presence or absence of 10 ng/ml of 11-KT. Late type B spermatogonia (proliferated spermatogonia) were observed in treatments of 100 ng/ml of both rhIGF-I and -II in combination with 11-KT, indicating the onset and progression of spermatogenesis. In all tested rhIGF-I concentrations (except 0.1 ng/ml) supplemented with 11-KT, late type 8 spermatogonia were detected in at least one individual. Then, we proceeded with an in vitro 45-day culture of testicular tissue with 100 ng/ml of rhIGF-I in the presence or absence of 10 ng/ml of 11-KT to test the long-term effects of rhIGF-I on the spermatogenetic cycle. The presence of all types of germ cells, including spermatozoa, in the testis cultured with the admixture of the two hormones indicated that the germ cells underwent complete spermatogenesis whereas no germ cell proliferation was observed when the rhIGF-I was applied alone. These results suggest that IGF-I in the presence of 11-KT plays an essential role in the onset, progress, and regulation of spermatogenesis in the testis of the Japanese eel.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

In this study, we examined the in vitro effects of insulin-like growth factor I (IGF-I) in the presence or absence of 11-keto-testosterone (11-KT: the spermatogenesis-inducing hormone) on the proliferation of Japanese eel (Anguilla japonica) testicular germ cells. Initially, a short-term culture (15 days) of testicular tissue with only type A and early type B spermatogonia (preproliferated spermatogonia) was carried out in Leibovitz-15 growth medium supplemented with different concentrations of recombinant human IGF (rhIGF)-I or -II in the presence or absence of 10 ng/ml of 11-KT. Late type B spermatogonia (proliferated spermatogonia) were observed in treatments of 100 ng/ml of both rhIGF-I and -II in combination with 11-KT, indicating the onset and progression of spermatogenesis. In all tested rhIGF-I concentrations (except 0.1 ng/ml) supplemented with 11-KT, late type 8 spermatogonia were detected in at least one individual. Then, we proceeded with an in vitro 45-day culture of testicular tissue with 100 ng/ml of rhIGF-I in the presence or absence of 10 ng/ml of 11-KT to test the long-term effects of rhIGF-I on the spermatogenetic cycle. The presence of all types of germ cells, including spermatozoa, in the testis cultured with the admixture of the two hormones indicated that the germ cells underwent complete spermatogenesis whereas no germ cell proliferation was observed when the rhIGF-I was applied alone. These results suggest that IGF-I in the presence of 11-KT plays an essential role in the onset, progress, and regulation of spermatogenesis in the testis of the Japanese eel.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC  

In this work, we examined the functions of the female hormone "estrogen" on spermatogenesis of the Japanese eel (Anguilla japonica). Estradiol-17 beta (E-2), a natural estrogen in vertebrates, was present in the serum and its receptor was expressed in the testis during the whole process of spermatogenesis, Spermatogonial stem cell renewal was promoted by E-2 implantation but was suppressed by tamoxifen (an antagonist of estrogen), In vitro, 10 pg/ml of E-2 was sufficient to induce spermatogonial stem cell division in cultured testicular tissue, therefore confirming the in vivo observations. These results clearly show that estrogen is an indispensable "male hormone" in the early spermatogenetic cycle. (C) 1999 Academic Press.

DOI： 10.1006/bbrc.1999.1494

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE ASIA  

A single injection of human chorionic gonadotropin (HCG) can induce complete spermatogenesis in immature Japanese eel (Anguilla japonica) testes consisting of only premitotic spermatogonia. Proliferation of spermatogonia, meiosis and spermiogenesis begin on 3, 12 and 18 days after HCG injection, respectively. To isolate the genes responsible for regulating the initiation of meiosis, differential mRNA display using poly (A)(+) RNA extracted from testes of eels at different times after HCG treatment was carried out. Five cDNA clones in which expression was initiated before the onset of meiosis were obtained. Northern blot analysis showed that one clone, which encoded activin PB subunit, was expressed in the initial phase of spermatogenesis (1-6 days after HCG treatment), in agreement with the previous suggestion that activin B induces the initiation of spermatogenesis in the Japanese eel. The remaining four were expressed in the testes during the following time frames. 3-18 days (two clones), 6-18 days (one clone) and 9-18 days (one clone) after HCG treatment. One of the two clones expressed on day 3 exhibited strong expression on days 12 and 15, just at the initiation period of meiosis. This clone was selected as a candidate gene responsible for initiating meiosis, and its full-length cDNA isolated. The cDNA contained an open reading frame of 1571 nucleotides encoding a protein of 260 amino acid residues, which showed high homology with the proliferating cell nuclear antigen (PCNA) of human, mouse and Xenopus. Northern blot analysis using eel PCNA cDNA showed that a 1.6 kb transcript first appeared on day 3 and became abundant, reaching maximum levels on days 12-15. In situ hybridization analysis revealed that PCNA mRNA was expressed strongly in late type B spermatogonia before the sixth mitotic division. it has already been shown that spermatogonia have a regulatory point to enter meiosis between the fifth and sixth mitotic division. The coincidence of PCNA expression and this regulatory point suggests an involvement of PCNA in the progression of mitotic germ cells into meiosis during HCG-induced spermatogenesis in the eel.

DOI： 10.1046/j.1440-169X.1999.00445.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE ASIA  

A single injection of human chorionic gonadotropin (HCG) can induce complete spermatogenesis in immature Japanese eel (Anguilla japonica) testes consisting of only premitotic spermatogonia. Proliferation of spermatogonia, meiosis and spermiogenesis begin on 3, 12 and 18 days after HCG injection, respectively. To isolate the genes responsible for regulating the initiation of meiosis, differential mRNA display using poly (A)(+) RNA extracted from testes of eels at different times after HCG treatment was carried out. Five cDNA clones in which expression was initiated before the onset of meiosis were obtained. Northern blot analysis showed that one clone, which encoded activin PB subunit, was expressed in the initial phase of spermatogenesis (1-6 days after HCG treatment), in agreement with the previous suggestion that activin B induces the initiation of spermatogenesis in the Japanese eel. The remaining four were expressed in the testes during the following time frames. 3-18 days (two clones), 6-18 days (one clone) and 9-18 days (one clone) after HCG treatment. One of the two clones expressed on day 3 exhibited strong expression on days 12 and 15, just at the initiation period of meiosis. This clone was selected as a candidate gene responsible for initiating meiosis, and its full-length cDNA isolated. The cDNA contained an open reading frame of 1571 nucleotides encoding a protein of 260 amino acid residues, which showed high homology with the proliferating cell nuclear antigen (PCNA) of human, mouse and Xenopus. Northern blot analysis using eel PCNA cDNA showed that a 1.6 kb transcript first appeared on day 3 and became abundant, reaching maximum levels on days 12-15. In situ hybridization analysis revealed that PCNA mRNA was expressed strongly in late type B spermatogonia before the sixth mitotic division. it has already been shown that spermatogonia have a regulatory point to enter meiosis between the fifth and sixth mitotic division. The coincidence of PCNA expression and this regulatory point suggests an involvement of PCNA in the progression of mitotic germ cells into meiosis during HCG-induced spermatogenesis in the eel.

DOI： 10.1046/j.1440-169x.1999.00445.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Ionic composition of the seminal plasma and factors that initiate sperm motility in the freshwater Asian catfish Clarias macrocephalus, were examined to develop an artificial seminal plasma (ASP) that can be used to dilute milt, The optimum ratio of milt:ASP that can reversibly activate the sperm and milt-ASP:ovulated eggs that will result in high fertilization rates were further determined to minimize the number of males to be sacrificed during artificial insemination. Seminal plasma of C, macrocephalus contained 17.8 +/- 0.1 mM/l potassium, 164.4 +/- 0.6 nM/l sodium, 8.4 +/- 0.0 mM/l calcium and 1.6 +/- 0.0 mM/l magnesium, and had an osmolality of 269.0 +/- 6.4 mOsm/kg, and pH of 7.8 +/- 0.2. Sperm motility was highest and longest in all electrolyte (NaCl, CaCl2,, KCl) and non-electrolyte (mannitol) solutions of 200 mOsm/kg. Catfish sperm were motile in all isotonic NaCl-KCl solutions, and were reversibly activated in the ASP (143 mM NaCl, 30 mM KCl, 8 mM CaCl2, 2 mM MgCl2, 10 mM HERES) solutions of pH 6.4-9.4. Altogether, these results suggest that sperm motility in C. macrocephalus was mainly initiated by a decrease in osmotic pressure, rather than ions and pH. High fertilization rates (89-94%) were observed when 10 mu l milt, diluted with 1000 mu l ASP, was activated with 5 mi of 0.6% NaCl (198.24 mOsm/kg) to fertilize 5 or 10 g of ovulated eggs, Results obtained from the present study provide information on sperm physiology that will lead to mon efficient gamete management, and hopefully, an increase in the yield of catfish fry in the hatchery. (C) 1999 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0044-8486(98)00402-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

A single injection of human chorionic gonadotropin (HCG) can induce complete spermatogenesis in immature eel testes consisting of premitotic spermatogonia. To understand the regulatory mechanisms of spermatogenesis, we have applied a subtractive hybridization method to identify genes in which changes in expression occur after HCG treatment in vivo. The subtraction was carried out 24 hours after HCG injection. Two up-regulated and six down regulated cDNA clones by HCG stimulation were isolated, and named eel spermatogenesis-related substance (eSRS) 1 to 8. In this paper, down-regulated cDNA clones of eSRS3 and eSRS4 were sequenced. A homology search showed that eSRS3 and eSRS4 have amino acid sequences similar to those of the ZP-domains of zona pellucida sperm-binding protein (ZP)-2 and 3, respectively. Transcripts of eSRS3 and eSRS4 have been detected only in immature testes and ovaries. Both transcripts disappeared immediately after HCG injection and were not detected in testes throughout the experimental period. To determine whether HCG action on down-regulation of eSRS3 and eSRS4 transcription is direct or mediated through 11-ketotestosterone (11-KT), a spermatogenesis-inducing steroid in eel, we investigated the effect of HCG and 11-KT on testicular eSRS3 and eSRS4 mRNA transcription in vitro. Northern blot analysis using poly(A)(+) RNA extracted from cultured testis showed that both HCG and 11-KT suppressed the mRNA transcription of both eSRS3 and eSRS4. We speculate that eSRS3 and eSRS4 may play important roles in the prevention of spermatogenesis in the eel. (C) 1998 Wiley-Liss, Inc.

DOI： 10.1002/(SICI)1098-2795(199811)51:3<235::AID-MRD2>3.0.CO;2-Q

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC SCI FISHERIES TOKYO UNIV FISHERIES  

Testicular development, gonadosomatic index (GSI), and related steroid hormones (testosterone or T, 11-ketotestosterone or 11-KT, 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one or DHP) in serum were monitored during an annual reproductive cycle in tank-reared, hatchery-bred male catfish Clarias macrocephalus to establish the season optimum for its artificial propagation. GSI values were highest in June (0.80%), and lowest in December, February, April (0.36%). At most times of the year, lobules in the testis and seminal vesicles were mostly lined with spermatogonia B (SGb) and spermatocytes (SC) and few spermatogonia A (SGa); spermatids (SD) and spermatozoa (SZ) were the least and most abundant of the spermatogenic cells, respectively. In January however, almost equal counts of SGa, SGb and SC were observed, as well as a significant increase in the percentage of SD and a corresponding decrease in SZ. Serum 11-KT fluctuated at high levels, with the lowest level in January (159.42 ng/ml), and peak in September (434.72 ng/ml). Serum T levels ranged from 15-25 ng/ml, and were not markedly different throughout the annual cycle. Serum DHP levels were extremely low in January-May, and reached maximum levels in July (0.18 ng/ml). Seasonal changes in the percentage of spermatogenic cells, GSI and serum steroid hormone profiles showed that captive, hatchery-bred male C. macrocephalus have a continuous reproductive cycle. Although milt release was not observed, males can readily be used as source of milt for artificial propagation at any time of the annual cycle, except in January.

DOI： 10.2331/fishsci.63.681

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KLUWER ACADEMIC PUBL  

The initial appearance and the development of Leydig cells (LCs), the sites of steroid hormone production in the testis, were investigated ultrastructurally during testicular differentiation in the Japanese eel, Anguilla japonica. In addition, the effects of a single injection of human chorionic gonadotropin (HCG; 5 IU g body weight(-1)) on histological changes of the testes and serum 11-ketotestosterone (11-KT) were examined at various stages (15-18, 20-23, 26-29, 32-35, 38-41 and 46-50 cm body length (BL)) of testicular differentiation. Testicular differentiation was morphologically characterized by the development of loose connective tissue on the medial side in animals 18-29 cm in BL. Ultrastructurally, LCs were first identified in the loose connective tissue of the testis of the 23 cm fish. In the testes of fish over 32 cm, clusters of LCs were distributed throughout the interstitial region accompanying the increase in number of spermatogonia. In fish larger than 32 cm, spermatogenesis was induced by administration of HCG; serum 11-KT levels were also raised. On the other hand, there was no effect on spermatogenesis or serum 11-KT levels in fish less than 29 cm, or in the controls. These result suggests that morphological differentiation of LCs occurs in testis of the 23 cm eel, and subsequently, the testes of eels of BL more than 32 cm acquire the capability to produce steroid hormones.

DOI： 10.1023/A:1007753115397

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The initial appearance and the development of Leydig cells (LCs), the sites of steroid hormone production in the testis, were investigated ultrastructurally during testicular differentiation in the Japanese eel, Anguilla japonica. In addition, the effects of a single injection of human chorionic gonadotropin (HCG; 5 IU g body weight-1) on histological changes of the testes and serum 11-ketotestosterone (11-KT) were examined at various stages (15-18, 20-23, 26-29, 32-35, 38-41 and 46-50 cm body length (BL)) of testicular differentiation. Testicular differentiation was morphologically characterized by the development of loose connective tissue on the medial side in animals 18-29 cm in BL. Ultrastructurally, LCs were first identified in the loose connective tissue of the testis of the 23 cm fish. In the testes of fish over 32 cm, clusters of LCs were distributed throughout the interstitial region accompanying the increase in number of spermatogonia. In fish larger than 32 cm, spermatogenesis was induced by administration of HCG; serum 11-KT levels were also raised. On the other hand, there was no effect on spermatogenesis or serum 11-KT levels in fish less than 29 cm, or in the controls. These result suggests that morphological differentiation of LCs occurs in testis of the 23 cm eel, and subsequently, the testes of eels of BL more than 32 cm acquire the capability to produce steroid hormones.

DOI： 10.1023/A:1007753115397

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BioScientifica Ltd.  

We have cloned a full length cDNA coding for activin β(B) subunit from the goldfish ovary. Sequence analysis of the goldfish activin β(B) shows that this peptide is extremely conserved across vertebrates. The mature region of goldfish activin β(B) has 93 and 98% amino acid identity with that of human and zebrafish β(B) subunit respectively. The identity of the cloned goldfish activin β(B) was further confirmed by expressing the protein in the Chinese hamster ovary (CHO) cells followed by detection of the specific activity of activin in the culture medium using F5-5 cell assay. mRNA of goldfish activin β(B) is expressed in a variety of goldfish tissues including ovary, testis, brain, pituitary, kidney and liver, suggesting a wide range of physiological roles for activin in the goldfish.

DOI： 10.1677/jme.0.0190037

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Blackwell Publishing  

In the cultivated male Japanese eel, spermatogonia are the only germ cells present in the testis. Weekly injections of human chorionic gonadotropin (HCG) can induce complete spermatogenesis from proliferation of spermatogonia to spermiogenesis. In some cases, however, HCG injection fails to induce complete spermatogenesis. Testicular morphological observations that HCG-injected eels could be classified into three types based On their testicular conditions. Type 1 eels had a well-developed testis and the milt could be acquired by hand-stripping. In type 2 eels, spermatogenesis was also induced by HCG injection, but testicular size was remarkably smaller than that of type 1 eels, and the milt could not be hand-stripped. At the end of the experiment, type 2 fish had only spermatogonia and a small amount of spermatozoa, but no spermatocytes or spermatids, in their testis. Type 3 eels had thready testis, which did not develop any germ cells during the experimental period. These results suggest that, despite elevations of plasma 11-ketotestosterone levels, HCG injections were not successful in inducing the completion of spermatogenesis in type 2 and type 3 eels. In most spermatogonia of type 2 eels, meiosis was not induced bY HCG injections. Furthermore, only few mitotic divisions had occurred as evidenced by the presence to 23 to 26 late type B spermatogonia in most cysts. This suggests that spermatogonial stem cells undergo four or five, and occasionally six, mitotic divisions before the interruption of spermatogenesis in type 2 eels. It is proposed that those numbers of mitotic divisions are related to a mediator that regulates entry of spermatogonia of the Japanese eel into meiosis.

DOI： 10.1046/j.1440-169X.1997.t01-5-00004.x

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Language：Japanese   Publisher：JAPAN SOCIETY FOR COMPARATIVE ENDOCRINOLOGY  

DOI： 10.5983/nl2001jsce.22.83_29

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE  

In cultivated male eel, spermatogonia are the only germ cells present in testis. Our previous studies using an organ culture system have shown that gonadotropin and 11-ketotestosterone (11-KT, a potent androgen in teleost fishes) can induce all stages of spermatogenesis in vitro. For detailed investigation of the control mechanisms of spermatogenesis, especially of the interaction between germ cells and testicular somatic cells during 11-KT-induced spermatogenesis in vitro, we have established a new culture system in which germ cells and somatic cells are cocultured after they are aggregated into pellets by centrifugation. Germ cells (spermatogonia) and somatic cells (mainly Sertoli cells) were isolated from immature eel testis. Coculture of the isolated germ cells and somatic cells without forming aggregation did not induce spermatogenesis, even in the presence of 11-KT. In contrast, when isolated germ cells and somatic cells were formed into pellets by centrifugation and were then cultured with 11-KT for 30 days, the entire process of spermatogenesis from premitotic spermatogonia to spermatozoa was induced. However, in the absence of 11-KT in the culture medium spermatogenesis was not induced, even when germ cell and somatic cells were aggregated. These results demonstrate that physical contact of germ cells to Sertoli cells is required for inducing spermatogenesis in response to 11-KT.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Blackwell Publishing  

In cultivated male eel, spermatogonia are the only germ cells present in testis. Our previous studies using an organ culture system have shown that gonadotropin and 11-ketotestosterone (11-KT, a potent androgen in teleost fishes) can induce all stages of spermatogenesis in vitro. For detailed investigation of the control mechanisms of spermatogenesis, especially of the interaction between germ cells and testicular somatic cells during 11-KT- induced spermatogenesis in vitro, we have established a new culture system in which germ cells and somatic cells are cocultured after they are aggregated into pellets by centrifugation. Germ cells (spermatogonia) and somatic cells (mainly Sertoli cells) were isolated from immature eel testis. Coculture of the isolated germ cells and somatic cells without forming aggregation did not induce spermatogenesis, even in the presence of 11-KT. In contrast, when isolated germ cells and somatic cells were formed into pellets by centrifugation and were then cultured with 11-KT for 30 days, the entire process of spermatogenesis from premitotic spermatogonia to spermatozoa was induced. However, in the absence of 11-KT in the culture medium spermatogenesis was not induced, even when germ cell and somatic cells were aggregated. These results demonstrate that physical contact of germ cells to Sertoli cells is required for inducing spermatogenesis in response to 11-KT.

DOI： 10.1046/j.1440-169X.1996.t01-2-00004.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INDIAN ACADEMY SCIENCES  

The testis of Japanese eel (Anguilla japonica) consists of type A and early type B spermatogonia together with inactive Leydig and Sertoli cells. A single injection of human chorionic gonadotropin induced marked changes in the morphology of the testis and in the serum androgen levels within a period of 72 h. Morphological changes include spermatogonial proliferation, activation of Leydig and Sertoli cells, organization of seminiferous lobules and formation of lobular lumen in the testis. Leydig cells were enlarged, exhibiting characteristics of steroid-producing cells. Sertoli cells become elongated, show signs of high cellular activity and remain in close contact with spermatogonia. The lobular organization was achieved much earlier than the progression of spermatogenesis to late type B spermatogonia Even 6 h after hCG injection, a significant increase in plasma levels of 11-ketotestosterone was observed, followed by a further time dependent increase. Plasma testosterone levels were also increased after injection, but the increase was much less than that of 11-ketotestosterone.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INDIAN ACADEMY SCIENCES  

The testis of Japanese eel (Anguilla japonica) consists of type A and early type B spermatogonia together with inactive Leydig and Sertoli cells. A single injection of human chorionic gonadotropin induced marked changes in the morphology of the testis and in the serum androgen levels within a period of 72 h. Morphological changes include spermatogonial proliferation, activation of Leydig and Sertoli cells, organization of seminiferous lobules and formation of lobular lumen in the testis. Leydig cells were enlarged, exhibiting characteristics of steroid-producing cells. Sertoli cells become elongated, show signs of high cellular activity and remain in close contact with spermatogonia. The lobular organization was achieved much earlier than the progression of spermatogenesis to late type B spermatogonia Even 6 h after hCG injection, a significant increase in plasma levels of 11-ketotestosterone was observed, followed by a further time dependent increase. Plasma testosterone levels were also increased after injection, but the increase was much less than that of 11-ketotestosterone.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INDIAN ACADEMY SCIENCES  

The testis of Japanese eel (Anguilla japonica) consists of type A and early type B spermatogonia together with inactive Leydig and Sertoli cells. A single injection of human chorionic gonadotropin induced marked changes in the morphology of the testis and in the serum androgen levels within a period of 72 h. Morphological changes include spermatogonial proliferation, activation of Leydig and Sertoli cells, organization of seminiferous lobules and formation of lobular lumen in the testis. Leydig cells were enlarged, exhibiting characteristics of steroid-producing cells. Sertoli cells become elongated, show signs of high cellular activity and remain in close contact with spermatogonia. The lobular organization was achieved much earlier than the progression of spermatogenesis to late type B spermatogonia Even 6 h after hCG injection, a significant increase in plasma levels of 11-ketotestosterone was observed, followed by a further time dependent increase. Plasma testosterone levels were also increased after injection, but the increase was much less than that of 11-ketotestosterone.

DOI： 10.1007/BF02703307

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.2331/fishsci.61.533

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Activin, a member of the transforming growth factor-β superfamily, has recently been isolated from eel testicular cDNA library. Using an in vitro testicular organ culture system, we investigated the effect of human recombinant activin A and B in eel spermatogenesis. Addition of human recombinant activin A and B (10 ng and 100 ng/ml) to the culture medium induced proliferation of spermatogonia within 15 days in the same manner as addition of 11-ketotestosterone (10 ng/ml) for positive control. In cultured testes without activins, however, the proliferation of spermatogonia was not observed. Although it is necessary to investigate the eel's own activin A and B protein, these results suggest the possibility that activin induces initiation of spermatogenesis in Japanese eel. © 1995, The Japanese Society of Fisheries Science. All rights reserved.

DOI： 10.2331/fishsci.61.434

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KUGLER PUBLICATIONS BV  

Changes in gonadal and plasma concentrations of thyroid hormones were examined at various stages of maturation in chum salmon (Oncorhynchus keta) caught in the Bering Sea and the Bay of Alaska. Plasma concentrations of thyroxine (T4) were less than 5 ng ml-1, and those of 3,5,3'-triiodo-L-thyronine (T3) were less than 2 ng ml-1 in both males and females, regardless of the degree of sexual maturity or the gonadosomatic index (GSI).
There was no clear relationships between circulating thyroid hormone levels and tissue levels. The ovarian T4 concentrations were undetectable (less than 0.2 ng g-1) or less than 2 ng g-1 when GSI was lower than 1%, but increased thereafter and reached a plateau of 8- 1 0 ng g-1 when GSI became 2%. The ovarian T3 concentrations were about 5 ng g-1 when GSI was 1%, increased to a maximum level (20 ng g-1) when GSI was about 2%, and decreased to a constant level of 10 ng g-1 thereafter. The T4 and T3 content in single oocyte increased proportionally to the oocyte volume, indicating a constant incorporation of the hormones into the oocyte.
The T4 concentrations in the testis were 1 ng g-1 or less regardless of the GSI. On the other hand, the T3 concentrations were highest (15 ng g-1) when the GSI was less than 1%, decreased thereafter when spermatocytes appeared in the testis, and became about 5 ng g-1 in testes containing spermatozoa, raising the possibility of a role for T3 during early gamete and/or gonad maturation of testes.

DOI： 10.1007/BF00004361

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KUGLER PUBLICATIONS BV  

Changes in gonadal and plasma concentrations of thyroid hormones were examined at various stages of maturation in chum salmon (Oncorhynchus keta) caught in the Bering Sea and the Bay of Alaska. Plasma concentrations of thyroxine (T4) were less than 5 ng ml-1, and those of 3,5,3'-triiodo-L-thyronine (T3) were less than 2 ng ml-1 in both males and females, regardless of the degree of sexual maturity or the gonadosomatic index (GSI).
There was no clear relationships between circulating thyroid hormone levels and tissue levels. The ovarian T4 concentrations were undetectable (less than 0.2 ng g-1) or less than 2 ng g-1 when GSI was lower than 1%, but increased thereafter and reached a plateau of 8- 1 0 ng g-1 when GSI became 2%. The ovarian T3 concentrations were about 5 ng g-1 when GSI was 1%, increased to a maximum level (20 ng g-1) when GSI was about 2%, and decreased to a constant level of 10 ng g-1 thereafter. The T4 and T3 content in single oocyte increased proportionally to the oocyte volume, indicating a constant incorporation of the hormones into the oocyte.
The T4 concentrations in the testis were 1 ng g-1 or less regardless of the GSI. On the other hand, the T3 concentrations were highest (15 ng g-1) when the GSI was less than 1%, decreased thereafter when spermatocytes appeared in the testis, and became about 5 ng g-1 in testes containing spermatozoa, raising the possibility of a role for T3 during early gamete and/or gonad maturation of testes.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KUGLER PUBLICATIONS BV  

Changes in gonadal and plasma concentrations of thyroid hormones were examined at various stages of maturation in chum salmon (Oncorhynchus keta) caught in the Bering Sea and the Bay of Alaska. Plasma concentrations of thyroxine (T4) were less than 5 ng ml-1, and those of 3,5,3'-triiodo-L-thyronine (T3) were less than 2 ng ml-1 in both males and females, regardless of the degree of sexual maturity or the gonadosomatic index (GSI).
There was no clear relationships between circulating thyroid hormone levels and tissue levels. The ovarian T4 concentrations were undetectable (less than 0.2 ng g-1) or less than 2 ng g-1 when GSI was lower than 1%, but increased thereafter and reached a plateau of 8- 1 0 ng g-1 when GSI became 2%. The ovarian T3 concentrations were about 5 ng g-1 when GSI was 1%, increased to a maximum level (20 ng g-1) when GSI was about 2%, and decreased to a constant level of 10 ng g-1 thereafter. The T4 and T3 content in single oocyte increased proportionally to the oocyte volume, indicating a constant incorporation of the hormones into the oocyte.
The T4 concentrations in the testis were 1 ng g-1 or less regardless of the GSI. On the other hand, the T3 concentrations were highest (15 ng g-1) when the GSI was less than 1%, decreased thereafter when spermatocytes appeared in the testis, and became about 5 ng g-1 in testes containing spermatozoa, raising the possibility of a role for T3 during early gamete and/or gonad maturation of testes.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

In salmonid fish, spermatozoa taken from the testes are immotile, but acquire motility during their passage through the sperm duct. Using male masu salmon (Oncorhynchus masou), we found that gonadotropin-induced testicular production of 17-alpha,20-beta-dihydroxy-4-pregnen-3-one (17-alpha,20-beta-DP), the oocyte maturation-inducing hormone of salmonid fish, is responsible for the acquisition of sperm motility. However, neither testosterone (T) nor 11-ketotestosterone (11-KT), the two major androgens in teleost fish, were effective. We also present evidence that 17-alpha,20-beta-DP action is mediated through an increase in sperm duct pH, which in turn increases the cAMP content of sperm allowing the acquisition of motility.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

In salmonid fish, spermatozoa taken from the testes are immotile, but acquire motility during their passage through the sperm duct. Using male masu salmon (Oncorhynchus masou), we found that gonadotropin-induced testicular production of 17-alpha,20-beta-dihydroxy-4-pregnen-3-one (17-alpha,20-beta-DP), the oocyte maturation-inducing hormone of salmonid fish, is responsible for the acquisition of sperm motility. However, neither testosterone (T) nor 11-ketotestosterone (11-KT), the two major androgens in teleost fish, were effective. We also present evidence that 17-alpha,20-beta-DP action is mediated through an increase in sperm duct pH, which in turn increases the cAMP content of sperm allowing the acquisition of motility.

DOI： 10.1002/jez.1402610316

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Language：English   Publishing type：Research paper (scientific journal)  

In salmonid fish, spermatozoa taken from the testes are immotile, but acquire motility during their passage through the sperm duct. Using male masu salmon (Oncorhynchus masou), we found that gonadotropin‐induced testicular production of 17α,20β‐dihydroxy‐4‐pregnen‐3‐one (17α,20β‐DP), the oocyte maturation‐inducing hormone of salmonid fish, is responsible for the acquisition of sperm motility. However, neither testosterone (T) nor 11‐ketotestosterone (11‐KT), the two major androgens in teleost fish, were effective. We also present evidence that 17α,20β‐DP action is mediated through an increase in sperm duct pH, which in turn increases the cAMP content of sperm allowing the acquisition of motility. Copyright © 1992 Wiley‐Liss, Inc., A Wiley Company

DOI： 10.1002/jez.1402610316

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED RES FOUND  

In the cultivated male Japanese eel, spermatogonia are the only germ cells present in the testis. Five weekly injections of human chorionic gonadotropin (HCG) induced spermatogenesis and spermiogenesis. This was accompained by a marked increase in serum levels of 11-ketotestosterone and testosterone; the increase in the former was much greater than that of the latter. These results suggest that the action of HCG in inducing testicular development in male eel is mediated by the production of 11-ketotestosterone in the testis. Despite the appearance of spermatozoa in the testis, HCG injections produced very little milt. Serum levels of 17-alpha,20-beta-dihydroxy-4-pregnen-3-one (17-alpha,20-beta-DP), a steroid which has been shown to be effective in inducing oocyte maturation and spermiation in several teleosts, remained very low throughout the period of HCG treatment. However, in vitro incubation studies showed that HCG-treated testes were able to convert exogenous 17-alpha-hydroxyprogesterone to 17-alpha,20-beta-DP, indicating that these testes possess 20-beta-hydroxysteroid dehydrogenase. Injections of 17-alpha,20-beta-DP to these males significantly raised milt volume and decreased spermatocrit values. 17-alpha,20-beta-DP injections also markedly increased the percentage of motile sperm and the duration of sperm motility, concomitant with a significant increase in the pH of the sperm duct, but not that of the testis. Taken together, these results suggest that 17-alpha,20-beta-DP plays an important role in inducing final testicular maturation and sperm motility in male eel, the latter action of the steroid being mediated probably through an increase in the sperm duct pH. It is concluded that incomplete testicular and sperm maturation by HCG injections is due in part to the failure of the testis to produce 17-alpha,20-beta-DP in response to HCG stimulation.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED RES FOUND  

In the cultivated male Japanese eel, spermatogonia are the only germ cells present in the testis. Five weekly injections of human chorionic gonadotropin (HCG) induced spermatogenesis and spermiogenesis. This was accompained by a marked increase in serum levels of 11-ketotestosterone and testosterone; the increase in the former was much greater than that of the latter. These results suggest that the action of HCG in inducing testicular development in male eel is mediated by the production of 11-ketotestosterone in the testis. Despite the appearance of spermatozoa in the testis, HCG injections produced very little milt. Serum levels of 17-alpha,20-beta-dihydroxy-4-pregnen-3-one (17-alpha,20-beta-DP), a steroid which has been shown to be effective in inducing oocyte maturation and spermiation in several teleosts, remained very low throughout the period of HCG treatment. However, in vitro incubation studies showed that HCG-treated testes were able to convert exogenous 17-alpha-hydroxyprogesterone to 17-alpha,20-beta-DP, indicating that these testes possess 20-beta-hydroxysteroid dehydrogenase. Injections of 17-alpha,20-beta-DP to these males significantly raised milt volume and decreased spermatocrit values. 17-alpha,20-beta-DP injections also markedly increased the percentage of motile sperm and the duration of sperm motility, concomitant with a significant increase in the pH of the sperm duct, but not that of the testis. Taken together, these results suggest that 17-alpha,20-beta-DP plays an important role in inducing final testicular maturation and sperm motility in male eel, the latter action of the steroid being mediated probably through an increase in the sperm duct pH. It is concluded that incomplete testicular and sperm maturation by HCG injections is due in part to the failure of the testis to produce 17-alpha,20-beta-DP in response to HCG stimulation.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

The importance of gonadotropins and androgens for spermatogenesis is generally accepted in vertebrates, but the role played by specific hormones has not been clarified. Under cultivation conditions, male Japanese eels (Anguilla japonica) have immature testes containing only premitotic spermatogonia, type A and early-type B spermatogonia. In the present study, a recently developed organ-culture system for eel testes was used to determine in vitro effects of various steroid hormones on spermatogenesis. After 9 days of culture in serum-free, chemically defined medium containing 11-ketotestosterone (10 ng/ml), a major androgen in male eels, type A and early-type B spermatogonia began mitosis, producing late-type B spermatogonia. After 18 days, zygotene spermatocytes with synaptonemal complexes appeared, indicating that meiosis had already started by this time. In testis fragments cultured for 21 days, round spermatids and spermatozoa were observed with spermatogenic cells at all stages of development. Addition of 11-ketotestosterone to the culture medium also caused a marked cytological activation of Sertoli cells. No other steroid hormones tested had such stimulatory effects. These results, together with our earlier observations, suggest the following sequence for the hormonal induction of spermatogenesis in eel testes; gonadotropin stimulates the Leydig cells to produce 11-ketotestosterone, which, in turn, activates the Sertoli cells leading to the completion of spermatogenesis. This is, thus, an example of an animal system in which all stages of spermatogenesis have been induced by hormonal manipulation in vitro.

DOI： 10.1073/pnas.88.13.5774

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Cultivated males of the Japanese eel (Anguilla japonica) were given a single injection of human chorionic gonadotropin (HCG; 5 IU/g BW), and histological changes in the testis were observed. Profiles of serum androgens (11-ketotestosterone and testosterone) and production of androgens by the testis in vitro were also measured. 1) Histology of the testis: Prior to HCG treatment, germ cells of male Japanese eel were all spermatogonia, and the morphology of Sertoli cells and Leydig cells indicated little activity. One day after injection, the first effect of HCG treatment was observed, consisting of the activation of Leydig and Sertoli cells. This was followed by proliferation of spermatogonia, which began after three days. After twelve days, some germ cells had begun meiosis. Spermatozoa were first observed after eighteen days. 2) Serum androgen profiles: Serum androgen levels were relatively low before HCG treatment, but had increased dramatically by one day after the treatment, and thereafter high levels were maintained throughout spermatogenesis. 3) In vitro production of androgens by the testis: The testis of uninjected eels produced androgens, principally 11-ketotestosterone, when cultured in vitro with HCG. Production was proportional to the concentration of HCG. These results indicate that a single injection of HCG can induce the proliferation of spermatogonia, the initiation of meiosis, and the induction of spermatogenesis. The phenomenon in the eel is associated with remarkable endocrinological changes, including the development of Leydig and Sertoli cells, and an increase in androgen production.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Cultivated males of the Japanese eel (Anguilla japonica) were given a single injection of human chorionic gonadotropin (HCG; 5 IU/g BW), and histological changes in the testis were observed. Profiles of serum androgens (11-ketotestosterone and testosterone) and production of androgens by the testis in vitro were also measured. 1) Histology of the testis: Prior to HCG treatment, germ cells of male Japanese eel were all spermatogonia, and the morphology of Sertoli cells and Leydig cells indicated little activity. One day after injection, the first effect of HCG treatment was observed, consisting of the activation of Leydig and Sertoli cells. This was followed by proliferation of spermatogonia, which began after three days. After twelve days, some germ cells had begun meiosis. Spermatozoa were first observed after eighteen days. 2) Serum androgen profiles: Serum androgen levels were relatively low before HCG treatment, but had increased dramatically by one day after the treatment, and thereafter high levels were maintained throughout spermatogenesis. 3) In vitro production of androgens by the testis: The testis of uninjected eels produced androgens, principally 11-ketotestosterone, when cultured in vitro with HCG. Production was proportional to the concentration of HCG. These results indicate that a single injection of HCG can induce the proliferation of spermatogonia, the initiation of meiosis, and the induction of spermatogenesis. The phenomenon in the eel is associated with remarkable endocrinological changes, including the development of Leydig and Sertoli cells, and an increase in androgen production.

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.2220/biomedres.12.241

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Language：Japanese   Publisher：北海道大学  

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Other Link： http://hdl.handle.net/2115/24072

Language：English   Publishing type：Research paper (scientific journal)  

In the cultivated male Japanese eel, spermatogonia are the only germ cells present in the testis. Using a newly developed organ culture system, we obtained evidence that human chorionic gonadotropin (HCG) can induce the entire process of spermatogenesis, in vitro, from spermatogonia to spermatozoa within 24 days. The HCG-induced spermatogenesis in vitro was accompanied by a marked activation of Sertoli cells and Leydig cells, occurring prior to the beginning of spermatogonial proliferation. These results indicate that gonadotropin triggers spermatogenesis in the Japanese eel and further suggest that this effect of gonadotropin is mediated through the actions of testicular somatic cells. © 1991.

DOI： 10.1016/0012-1606(91)90468-I

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Language：Japanese   Publisher：北海道立水産孵化場  

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Other Link： http://agriknowledge.affrc.go.jp/RN/2010391631

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Language：Japanese  

J-GLOBAL

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

CiNii Books

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

CiNii Books

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J-GLOBAL

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Language：English   Publisher：水産増殖談話会  

DOI： 10.11233/aquaculturesci.62.23

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J-GLOBAL

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

CiNii Books

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

CiNii Books

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J-GLOBAL

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)  

DOI： 10.5983/nl2008jsce.38.134

CiNii Books

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Other Link： https://jlc.jst.go.jp/DN/JALC/10008612273?from=CiNii

J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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Language：Japanese  

J-GLOBAL

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Language：Japanese  

J-GLOBAL

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Language：Japanese  

J-GLOBAL

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Language：Japanese  

J-GLOBAL

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Language：Japanese  

J-GLOBAL

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

CiNii Books

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

CiNii Books

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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Language：Japanese  

J-GLOBAL

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Language：Japanese  

J-GLOBAL

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Language：Japanese  

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Language：Japanese  

J-GLOBAL

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Language：English   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

DOI： 10.1016/j.ygcen.2010.02.001

Web of Science

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

CiNii Books

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J-GLOBAL

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J-GLOBAL

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (bulletin of university, research institution)  

CiNii Books

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Other Link： http://agriknowledge.affrc.go.jp/RN/2010770100

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：WILEY-LISS  

Web of Science

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：SOC STUDY REPRODUCTION  

Web of Science

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：SOC STUDY REPRODUCTION  

Web of Science

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J-GLOBAL

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Language：English   Publisher：Zoological Society of Japan  

CiNii Books

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Other Link： http://id.nii.ac.jp/1141/00037861/

Language：English   Publisher：Zoological Society of Japan  

CiNii Books

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Other Link： http://id.nii.ac.jp/1141/00036938/

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Language：English   Publisher：MEDIMOND S R L  

To understand the regulatory mechanisms of spermatogenesis in eel, we have applied a cDNA subtraction and differential display method to identify genes, which show unique expressions in eel testis during spermatogenesis. We have isolated 28 stage-specific cDNA clones and named them eel spermatogenesis related substances (eSRSs).
Spermatogenetic process from spermatogonial proliferation to spermiogenesis is controlled by 11-ketotestosterone (11-KT) through Sertoli cell action. Among the 28 eSRSs, the testicular mRNA expression of 16 types was changed by 11-KT treatment in vitro. Therefore, it is possible that some of these factors regulate spermatogenesis under 11-KT stimulation.

Web of Science

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Language：Japanese   Publisher：海洋出版  

CiNii Books

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Language：English   Publisher：Zoological Society of Japan  

CiNii Books

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Other Link： http://id.nii.ac.jp/1141/00034520/

Language：English   Publisher：Zoological Society of Japan  

CiNii Books

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Other Link： http://id.nii.ac.jp/1141/00034519/

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Language：English   Publisher：Zoological Society of Japan  

CiNii Books

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Other Link： http://id.nii.ac.jp/1141/00034478/

Language：English   Publisher：Zoological Society of Japan  

CiNii Books

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Other Link： http://id.nii.ac.jp/1141/00033658/

J-GLOBAL

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Language：Japanese   Publisher：Nihon Suisan Gakkai  

DOI： 10.2331/suisan.62.547

Scopus

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Language：English   Publisher：NATL RESEARCH COUNCIL CANADA  

Web of Science

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Language：English   Publisher：NATL RESEARCH COUNCIL CANADA  

Web of Science

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Language：English   Publishing type：Book review, literature introduction, etc.   Publisher：JOHN WILEY & SONS LTD  

Under cultivation conditions, male Japanese eels (Anguilla japonica) have immature testes containing only spermatogonia together with inactive testicular somatic cells, Leydig cells and Sertoli cells. Using a recently developed organ culture system for eel testes, we have shown that hormonal induction of spermatogenesis in eel testes involves gonadotropin stimulation of Leydig cells to produce 11-ketotestosterone, a potent androgen in fish. In turn, 11-ketotestosterone activates Sertoli cells to stimulate premitotic spermatogonia to complete spermatogenesis. Our current research focuses on the isolation and characterization of genes that show altered expression in eel testes during gonadotropin-induced spermatogenesis. One up-regulated and three down-regulated genes have been isolated. Northern blot analysis and in situ hybridization reveal that mRNA for activin B is absent in testes before gonadotropin injection and is abundant in Sertoli cells in testes injected with gonadotropin for one to six days after injection. This stimulation of activin B mRNA is accompanied by spermatogonial proliferation. Gonadotropin treatment also causes a rapid rise in the testicular concentrations of mRNA for 3 beta-hydroxysteroid dehydrogenase, the rate-limiting enzyme for gonadotropin-induced 11-ketotestosterone production. We have also obtained three downregulated cDNAs which are abundant in testes before gonadotropin treatment and disappear almost completely in testes one day after gonadotropin injection.

Web of Science

Scopus

PubMed

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J-GLOBAL

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Language：English   Publisher：Zoological Society of Japan  

CiNii Books

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Other Link： http://id.nii.ac.jp/1141/00029564/

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Applicant：株式会社愛南リベラシオ

Application no：特願2015-010883  Date applied：2015.1

Announcement no：特開2015-156856  Date announced：2015.9

J-GLOBAL

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Applicant：国立大学法人愛媛大学

Application no：JP2014073232  Date applied：2014.9

Announcement no：WO2015-033973  Date announced：2015.3

Publication no：WO2015-033973  Date published：2015.3

J-GLOBAL

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Applicant：国立大学法人愛媛大学

Application no：JP2014073231  Date applied：2014.9

Announcement no：WO2015-033972  Date announced：2015.3

Publication no：WO2015-033972  Date published：2015.3

J-GLOBAL

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Applicant：国立大学法人愛媛大学, 物産バイオテック株式会社

Application no：特願2014-011874  Date applied：2014.1

Announcement no：特開2015-137361  Date announced：2015.7

J-GLOBAL

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Applicant：株式会社愛南リベラシオ

Application no：JP2013069834  Date applied：2013.7

Announcement no：WO2014-017451  Date announced：2014.1

J-GLOBAL

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Applicant：株式会社愛南リベラシオ

Application no：特願2014-526915  Date applied：2013.7

Patent/Registration no：特許第6019505号  Date issued：2016.10

J-GLOBAL

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Applicant：三浦 猛, アビオス株式会社

Application no：特願2012-166000  Date applied：2012.7

Announcement no：特開2013-047220  Date announced：2013.3

J-GLOBAL

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Applicant：三浦 猛

Application no：特願2012-166001  Date applied：2012.7

Announcement no：特開2013-047221  Date announced：2013.3

J-GLOBAL

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Applicant：国立大学法人愛媛大学, 京葉プラントエンジニアリング株式会社, 株式会社三六九, 日環科学株式会社

Application no：特願2011-215795  Date applied：2011.9

Announcement no：特開2012-120526  Date announced：2012.6

J-GLOBAL

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Applicant：国立大学法人愛媛大学, 京葉プラントエンジニアリング株式会社, 株式会社三六九, 日環科学株式会社

Application no：特願2011-215795  Date applied：2011.9

Announcement no：特開2012-120526  Date announced：2012.6

Patent/Registration no：特許第6010725号  Date registered：2016.9  Date issued：2016.9

J-GLOBAL

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Applicant：国立大学法人愛媛大学

Application no：特願2011-000456  Date applied：2011.1

Announcement no：特開2012-130331  Date announced：2012.7

J-GLOBAL

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Applicant：国立大学法人愛媛大学, 愛媛県

Application no：特願2010-260076  Date applied：2010.11

Announcement no：特開2012-111698  Date announced：2012.6

J-GLOBAL

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Applicant：国立大学法人愛媛大学, アビオス株式会社

Application no：特願2011-522868  Date applied：2010.7

Patent/Registration no：特許第5759895号  Date registered：2015.6  Date issued：2015.6

J-GLOBAL

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Applicant：国立大学法人愛媛大学, アビオス株式会社

Application no：JP2010062055  Date applied：2010.7

Announcement no：WO2011-007867  Date announced：2011.1

Publication no：WO2011-007867  Date published：2011.1

Patent/Registration no：特許第5759895号  Date issued：2015.6

J-GLOBAL

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Applicant：国立大学法人愛媛大学

Application no：特願2009-036943  Date applied：2009.2

Announcement no：特開2009-219487  Date announced：2009.10

J-GLOBAL

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Applicant：国立大学法人愛媛大学

Application no：特願2007-066627  Date applied：2007.3

Announcement no：特開2007-284426  Date announced：2007.11

J-GLOBAL

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Applicant：国立大学法人愛媛大学

Application no：特願2006-338610  Date applied：2006.12

Announcement no：特開2008-148607  Date announced：2008.7

J-GLOBAL

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Applicant：三浦 猛

Application no：特願2006-256789  Date applied：2006.9

Announcement no：特開2008-074280  Date announced：2008.4

J-GLOBAL

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Applicant：日本水産株式会社

Application no：特願2005-356671  Date applied：2005.12

Announcement no：特開2006-306834  Date announced：2006.11

Patent/Registration no：特許第4976006号  Date issued：2012.4

J-GLOBAL

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Applicant：日本水産株式会社

Application no：特願2005-356671  Date applied：2005.12

Announcement no：特開2006-306834  Date announced：2006.11

J-GLOBAL

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