Nibea Mitsukurii Research Articles

Production of donor-derived eggs after ovarian germ cell transplantation into the gonads of adult, germ cell-less, triploid hybrid fish†.

In animals, spermatogonial transplantation in sterile adult males is widely developed; however, despite its utility, ovarian germ cell transplantation is not well developed. We previously showed that the interspecific hybrid offspring of sciaenid was a suitable model for germ cell transplantation studies as they have germ cell-less gonads. However, all these gonads have testis-like characteristics. Here, we tested whether triploidization in hybrid embryos could result in germ cell-less ovary development. Gonadal structure dimorphism and sex-specific gene expression patterns were examined in 6-month-old triploid hybrids (3nHybs). Thirty-one percent of 3nHybs had germ cell-less gonads with an ovarian cavity. cyp19a1a and foxl2, ovarian differentiation-related genes, were expressed in these gonads, whereas dmrt1 and vasa were not expressed, suggesting ovary-like germ cell-less gonad development. Some (26%) 3nHybs had testis-like germ cell-less gonads. Ovarian germ cells collected from homozygous green fluorescent protein (GFP) transgenic blue drum (BD) (Nibea mitsukurii) were transplanted into 6-month-old 3nHybs gonads via the urogenital papilla or oviduct. After 9months, the recipients were crossed with wild type BD. Among the six 3nHyb recipients that survived, one female and one male produced fertile eggs and motile sperm carrying gfp-specific DNA sequences. Progeny tests revealed that all F1 offspring possessed gfp-specific DNA sequences, suggesting that these recipients produced only donor-derived eggs or sperm. Histological observation confirmed donor-derived gametogenesis in the 3nHyb recipients' gonads. Overall, triploidization reduces male-biased sex differentiation in germ cell-less gonads. We report, for the first time, donor-derived egg production in an animal via direct ovarian germ cell transplantation into a germ cell-less ovary.

Induction of sex reversal in blue drum ( Nibea mitsukurii ) and gynogenetic yellow drum ( Nibea albiflora ) by oral administration of letrozole

The objective of this study was to test the efficiency of an aromatase inhibitor, letrozole (LZ), to induce masculinization in blue drum (Nibea mitsukurii) and gynogenetic yellow drum (N. albiflora). Two experiments (Exp-1 and Exp-2) were performed to investigate the effect of LZ on the growth, sex ratio and gonad development in these two fishes. In Exp-1, blue drum were treated with oral administration of letrozole at different doses (0, 1, 10, 100 mg/kg). In Exp-2, gynogenetic yellow drum were orally administered different doses of LZ (0, 10, 100 mg/kg) and a dose of 10 mg/kg 17α-methyltestosterone (MT). The treatments in both experiments were performed from 25 to 85 days post hatch (dph). As a result, all the LZ-treated fish were phenotypic males despite of slower growth than the control during the treatment period. Free spermatozoa were observed from 150 dph onwards and the gonadosomatic index of the LZ-treated fish did not differ significantly from that in the control at maturity. However, the sex-reversed fish induced by MT exhibited a high proportion (33.4%) of testicular abnormalities. Our results showed that oral administration of LZ was efficient in inducing sex reversal in drums, and thereby facilitated their mono-sex culture.

Germ cell-less hybrid fish: ideal recipient for spermatogonial transplantation for the rapid production of donor-derived sperm†.

An interspecific hybrid marine fish that developed a testis-like gonad without any germ cells, i.e., a germ cell-less gonad, was produced by hybridizing a female blue drum Nibea mitsukurii with a male white croaker Pennahia argentata. In this study, we evaluated the suitability of the germ cell-less fish as a recipient by transplanting donor testicular cells directly into the gonads through the urogenital papilla. The donor testicular cells were collected from hemizygous transgenic, green fluorescent protein (gfp) (+/-) blue drum, and transplanted into the germ cell-less gonads of the 6-month-old adult hybrid croakers. Fluorescent and histological observations showed the colonization, proliferation, and differentiation of transplanted spermatogonial cells in the gonads of hybrid croakers. The earliest production of spermatozoa in a hybrid recipient was observed at 7 weeks post-transplantation (pt), and 10% of the transplanted recipients produced donor-derived gfp-positive spermatozoa by 25 weeks pt. Sperm from the hybrid recipients were used to fertilize eggs from wild-type blue drums, and approximately 50% of the resulting offspring were gfp-positive, suggesting that all offspring originated from donor-derived sperm that were produced in the transplanted gfp (+/-) germ cells. To the best of our knowledge, this is the first report of successful spermatogonial transplantation using a germ cell-less adult fish as a recipient. This transplantation system has considerable advantages, such as the use of comparatively simple equipment and procedures, and rapid generation of donor-derived spermatogenesis and offspring, and presents numerous applications in commercial aquaculture.

Specific visualization of live type A spermatogonia of Pacific bluefin tuna using fluorescent dye-conjugated antibodies†.

During our previous work toward establishing surrogate broodstock that can produce donor-derived gametes by germ cell transplantation, we found that only type A spermatogonia (ASGs) have the potency to colonize recipient gonads. Therefore, the ability to visualize ASGs specifically would allow the sequential analysis of donor cell behavior in the recipient gonads. Here we produced monoclonal antibodies that could recognize the cell surface antigens of ASGs in Pacific bluefin tuna (Thunnus orientalis), with the aim of visualizing live ASGs. We generated monoclonal antibodies by inoculating Pacific bluefin tuna testicular cells containing ASGs into mice and then screened them using cell-based enzyme-linked immunosorbent assay (ELISA), immunocytochemistry, flow cytometry (FCM), and immunohistochemistry, which resulted in the selection of two antibodies (Nos. 152 and 180) from a pool of 1152 antibodies. We directly labeled these antibodies with fluorescent dye, which allowed ASG-like cells to be visualized in a one-step procedure using immunocytochemistry. Molecular marker analyses against the FCM-sorted fluorescent cells confirmed that ASGs were highly enriched in the antibody-positive fraction. To evaluate the migratory capability of the ASGs, we transplanted visualized cells into the peritoneal cavity of nibe croaker (Nibea mitsukurii) larvae. This resulted in incorporated fluorescent cells labeled with antibody No. 152 being detected in the recipient gonads, suggesting that the visualized ASGs possessed migratory and incorporation capabilities. Thus, the donor germ cell visualization method that was developed in this study will facilitate and simplify Pacific bluefin tuna germ cell transplantation.

Production of functionally sterile triploid Nibe croaker Nibea mitsukurii induced by cold-shock treatment with special emphasis on triploid aptitude as surrogate broodstock

Among the pelagic egg spawning marine teleosts, spermatogonial transplantation techniques were first developed in the Nibe croaker Nibea mitsukurii; however, production of donor-derived offspring had not yet been achieved. In salmonids, when triploid individuals were used as recipients, the resulting surrogate broodstock produced only donor-derived gametes, as the triploids were incapable of producing their own gametes. Therefore, in the present study, we determined the optimal parameters for cold-shock inducing triploidy through the suppression of meiosis II resulting in retention of the second polar body in fertilized Nibe croaker eggs. It was determined that cold-shock treatment, which was carried out at 10°C for a period of 15min beginning 5min after fertilization, following pre-shock treatment at 22°C achieved a 100% triploidization rate as well as hatching rates of which were high enough for practical application in the pelagic egg spawning marine teleost (16.4±10.4%). Average chromosome numbers of cold-shock treated larvae counted using mitotic figures found on the fin membrane of 1day post-hatch larvae were 67.3±0.7, which was approximately 1.5 times larger than that of control larvae (46.8±1.1), suggesting that triploidy had been induced. No differences in survival rates of hatched larvae during the rotifer-feeding stage existed between treated and non-treated groups. Gonadosomatic index and gametogenesis were impaired in both sexes of triploid Nibe croaker. Although triploids produced small amounts of gametes, no progeny from crosses between triploids and diploids survived later than 24h subsequent to hatching. These results indicated that the triploid Nibe croaker developed in this study could be effectively used as surrogate recipients in spermatogonial transplantation. Statement of relevanceIn intraperitoneal germ-cell transplantation techniques in salmonids, donor-derived diploid germ cells proliferated and differentiated within triploid recipient gonads, and the surrogate recipients produced only donor-derived gametes, which were capable of generating viable offspring when used in fertilization. To date, germ-cell transplantation techniques have been developed for use in perciforms, including sciaenids, however, triploid recipients have not yet been utilized in marine fishes. In this study, triploid Nibe croaker was produced at high triploidization rates (~100%) using cold-shock treatments. Analysis of the gonadal development and gametogenesis of triploid Nibe croaker in two consecutive spawning seasons revealed that the triploid Nibe croaker was functionally sterile in both sexes and that the triploids would be suitable for use as surrogate recipients.

Efficient production of donor-derived gametes from triploid recipients following intra-peritoneal germ cell transplantation into a marine teleost, Nibe croaker (Nibea mitsukurii)

Intra-peritoneal spermatogonial transplantation could become important in the production of allogenic or xenogenic donor-derived offspring from surrogate parent fish in commercial aquaculture. However, the rates at which surrogates with chimeric germ-lines are produced and contribute donor-derived gametes and offspring to subsequent generations must be improved for this technology to become practical, especially for marine fishes. Here, we performed intra-peritoneal spermatogonial transplantation into triploid Nibe croaker Nibea mitsukurii to evaluate the suitability of functionally sterile triploids as surrogates. Donor testicular cells were collected from hemizygous pHSC-GFP transgenic (gfp/−) individuals and then transplanted into the peritoneal cavities of 12-day-old larvae (diploid or triploid). By 6months after transplantation, 37% (male) and 29% (female) of triploid recipients had produced gfp-positive, donor-derived gametes; these rates were 7- and 4-fold higher, respectively, than the rates for diploid male and female recipients, respectively. Sperm and eggs from triploid recipients were artificially fertilized with eggs or sperm, respectively, from wild-type fish; approximately 50% of the resulting offspring were gfp-positive, suggesting that nearly all of offspring originated from donor-derived gametes that had descended from transplanted gfp/− germ cells. The fecundity and spawning frequency of surrogate triploid recipients were not significantly different to those of non-transplanted diploids, and the triploid recipients produced subsequent donor-derived offspring for at least two spawning seasons. Our results demonstrated that use of functionally sterile triploid recipients improved the percentage of recipients that produce donor-derived gametes. Further, the use of triploid recipients resulted in exclusive production of donor-derived gametes in this marine teleost. Statement of relevanceIntra-peritoneal spermatogonial transplantation using allogenic triploid recipients was first demonstrated in marine teleosts. In addition to efficient and exclusive donor-derived gametogenesis, we found that use of functionally sterile triploid recipients improved the percentage of recipients that produce donor-derived gametes. Consequently, triploid surrogates were more suitable than diploid surrogates and that use of triploid surrogates may accelerate the application of surrogate broodstock technology in aquaculture-targeted, genetically valuable, or endangered marine teleosts.

Polyunsaturated fatty acid metabolism in a marine teleost, Nibe croaker Nibea mitsukurii: Functional characterization of Fads2 desaturase and Elovl5 and Elovl4 elongases.

To reduce the requirement for fish oil in marine aquaculture, it would be advantageous to endow marine fish species with the capability for the endogenous biosynthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). For this purpose, we have previously produced transgenic Nibe croaker (Nibea mitsukurii) carrying an elongase of very-long-chain fatty acids 2 (elovl2) gene isolated from Masu salmon (Oncorhynchus masou). However, fatty acid analysis revealed that 24:5n-3 accumulated in the liver of the transgenic fish, whereas the DHA level did not differ between non-transgenic and transgenic fish. Therefore, to select more effective enzymes for successful transgenic synthesis of DHA, understanding the endogenous DHA biosynthetic pathway in the Nibe croaker is considered to be important. The present study aimed to investigate the biochemical functions of the Elovl5, Elovl4 and Fads2 enzymes involved in the DHA biosynthetic pathway in the Nibe croaker. The results showed that both Elovl5 and Elovl4 were able to elongate C18 fatty acids to C22 fatty acids and that Fads2 had Δ6 desaturase activity toward C18 fatty acids and weak Δ8 desaturase activity toward C20 fatty acids. On the other hand, Fads2 was found to lack the ability to convert 24:5n-3 to 24:6n-3, a fatty acid that can directly be converted to DHA via β-oxidation.

Transgenic modification of the n-3 HUFA biosynthetic pathway in nibe croaker larvae: improved DPA (docosapentaenoic acid; 22:5n-3) production

Marine fish are generally unable to produce sufficient quantities of n-3 highly unsaturated fatty acid (n-3 HUFA) such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3). Consequently, the seed production of marine fish requires careful nutritional enrichment of live feeds such as rotifers and brine shrimp Artemia to meet n-3 HUFA requirements for normal growth. Another strategy for improving n-3 HUFA availability is modifying the biosynthetic pathway of marine fish using transgenic technology. In this study, we conducted a feeding trial with non-transgenic and transgenic nibe croaker Nibea mitsukurii carrying the elongation of very long-chain fatty acids protein 2 (Elovl2) gene isolated from masu salmon Oncorhynchus masou and three groups of Artemia (non-enriched and enriched with two products). For all Artemia groups, docosapentaenoic acid (DPA, 22:5n-3), which is a direct product of Elovl2, was significantly higher in the transgenic fish than that in non-transgenic fish, despite the absence of DPA in all diets. Thus, applying transgenic techniques to marine fish at the larval stage are a powerful strategy for modifying n-3 HUFA biosynthetic pathways.

Improvement of ovulation induction by additive injection of 17,20β-Dihydroxy-4-pregnen-3-one after human chorionic gonadotropin administration in a pelagic egg spawning marine teleost, nibe croakerNibea mitsukurii(Jordan & Snyder)

This study aimed to develop the consistent ovulation induction method in a pelagic egg spawning marine teleost, nibe croaker Nibea mitsukurii. Attempts to induce oocyte maturation and ovulation in nibe croaker using human chorionic gonadotropin (hCG; 0.5 IU g−1) resulted in the normal progression of oocyte maturation and hydration, but a failure to induce ovulation in many individuals. This ovulation disorder was similarly observed even when the dose of hCG was increased 10 times (5 IU g−1) or decreased to one tenth (0.05 IU g−1), indicating that it cannot be completely overcome solely by hCG administration. However, this ovulation disorder could be completely overcome by subsequent administration of 17,20β-dihydroxy-4-pregnen-3-one (DHP) at the appropriate dose (0.5 μg g−1) and time (20 h after hCG administration). An increase in the number of individuals that ovulated due to DHP administration led to an increase in individuals producing larvae, resulting in an approximately threefold increase in the estimated number of larvae produced compared with the group of fish administered hCG alone. Thus, this ovulation induction method using DHP administration after hCG was demonstrated to overcome the ovulation disorder in nibe croaker and could be applicable to commercially important species with similar ovulation problems.

Biological Characteristics of Fish Germ Cells and their Application to Developmental Biotechnology

We have revealed several unique characteristics of germ cell development using rainbow trout, including the fact that spermatogonia transplanted into the peritoneal cavity of newly hatched embryos migrate toward recipient gonads, that spermatogonia transplanted into female recipients start oogenesis and produce functional eggs and that diploid germ cells transplanted into triploid trout can complete gametogenesis. By combining these unique features of fish germ cells, we established allogeneic and xenogeneic transplantation systems for spermatogonia in several fish species. Spermatogonia isolated from the mature testes of vasa-green fluorescent protein (Gfp) transgenic rainbow trout were transplanted into the peritoneal cavity of triploid masu salmon newly hatched embryos. These spermatogonia migrated toward recipient salmon genital ridges with extending pseudopodia and were subsequently incorporated into them. We further confirmed that the donor-derived spermatogonia resumed gametogenesis and produced sperm and eggs in male and female salmon recipients, respectively. By inseminating the resulting eggs and sperm, we obtained only rainbow trout offspring in the F1 generation, suggesting that the triploid salmon recipients produced functional gametes derived only from donor trout. We further confirmed that this intra-peritoneal transplantation of germ cells is applicable to several marine fishes, which could be of benefit in the production of bluefin tuna that has a large broodstock (>100 kg) and is difficult to maintain in captivity. Gamete production of bluefin tuna could be more easily achieved by generating a surrogate species, such as mackerel, that can produce tuna gametes.

Establishment of a stable transgenic strain in a pelagic egg spawning marine teleost, Nibe croaker Nibea mitsukurii

Genetic modification is an important technology for the global food production of foodstuffs. Transgenic fish strains can be established by microinjection, but this powerful method has not been developed in marine fishes due to the difficulties associated with handling their small and fragile pelagic eggs and larvae. The present paper reports the production of a transgenic strain of Nibe croaker Nibea mitsukurii (Sciaenidae), a marine fish that produces small, pelagic eggs. A microinjection technique was used to inject enhanced Green fluorescent protein ( eGfp) gene driven by rainbow trout heat-shock-cognate 71 ( hsc71) promoter/enhancer directly into fertilized eggs. Of 610 microinjected eggs, 498 (81.6%) survived to the 14-somite stage (24 h post-fertilization) and of these, 426 (69.8%) showed highly mosaic transient expression of GFP and were selected for rearing to sexual maturity. Of the surviving fish, one out of 44 males (2.2%) and 2 out of 22 females (9.1%) produced GFP-positive embryos. Between 1.4 and 10.5% F 1 embryos were GFP-positive. Both maternal and zygotic expression of the eGfp gene was observed in all F 1 transgenic offspring prior to hatching. Southern blot analysis and PCR analysis of the genomic DNA of F 2 offspring revealed that transgenes were integrated into the host genome and were transmitted in a Mendelian fashion. This is the first time that a transgenic strain has been established via the microinjection method in pelagic egg spawning marine fish. The establishment of genetic modification methods in marine aquaculture fishes would be a powerful tool for improving their commercially valuable traits by introducing genes that control a wide range of biological phenomena.

Colonization, proliferation, and survival of intraperitoneally transplanted yellowtail Seriola quinqueradiata spermatogonia in nibe croaker Nibea mitsukurii recipient

Recently, we developed an intraspecies spermatogonial transplantation technique in a pelagic egg spawning marine teleost, nibe croaker Nibea mitsukurii. Nibe croaker is an ideal candidate recipient for spermatogonial transplantation since it has a short generation time and small body size. In the present study, yellowtail Seriola quinqueradiata spermatogonia were transplanted into nibe croaker larvae, and the behavior of transplanted spermatogonia in recipient gonads was observed. Three weeks post-transplantation, yellowtail spermatogonia were incorporated into the gonads of 72 out of 88 recipients. An antiproliferating cell nuclear antigen was detected in incorporated yellowtail spermatogonia, suggesting that the xenogenic germ cells were proliferating in recipient gonads. Yellowtail vasa-positive spermatogonia survived for 11 months after transplantation in the gonads of recipient fish. Thus, we showed that the microenvironment in nibe croaker gonads can support the colonization, proliferation, and survival of germ cells derived from a different taxonomic family.

Degree of intraspecific genetic divergence and variability in three Sciaenid species

Abstrllct Genetic variations in Nibea mitsukurii, N. albijfora and Pennahia argentara from different localities were assayed electrophoretically. The genetic variability in N. mirsukurii was relatively high. According to the chi-square values for heterogeneity between two localities and genetic distance (D) values, N. albijfora from the Ariake Sea and the Inland Sea or the East China Sea (D=O.OO92 in average), and N. misukurii, from Tosa Bay vs, Atsumi Bay (D=~O.OO67) were considered to be independent subpopulations, The D value in N. albifiora from the Inland Sea VS, the East China Sea was quite small (D=O.OOlO), In P. argenrara populations, the D values were also small. Further, the relationship between genetic and geographic distances observed in the three species were discussed, P. argenrata showed a positive relationship between these two factors as compared to N. mirsukurii or N, albifiora. It seemed that the lower level of genetic divergence might be due to the frequent gene flow within the large-scale and wide distributional range of the population. During the past decade the application of gel electrophoresis coupled with selective enzyme stain­ ing has shown that a large amount genetic variation exists in natural populations of many species (Allen­ dorf and Utter, 1979). Phenotypes of allozyme can be scored during most stages of their life cycle and their genetics more easily interpreted than the pro­ portional and meristic characters employed in fish taxonomy. Genetic differences among populations within a defined body of water may conceivably decrease with time, due to gene flow by migration within sub­ populations. Alternatively, genetic differentiation could become· accentuated through time, perhaps because subpopulations become increasingly associ­ ated with discrete breeding areas (Felley and Avise, 1980). The fishes of the family Sciaenidae contribute sub­ stantially to the fisheries of the warm and shallow seas and estuaries of the world. Nibea mitsukurii (Jordan et Snyder) commonly known as nibe croak­ er, extends along the coast of the Pacific Ocean from Sendai Bay in the north to Hyuga Basin (an open bay) in the south. Nibea albiflora (Richardson), commonly known as roncader, occurs in Hong­ kong, the East China Sea and northwards to Port Arthur and southern Japan. Pennahia argentata (Houttuyn), commonly known as white croaker, is widely distributed in Japanese, Korean and Chinese waters (Trewavas, 1977).

Chub Mackerel Gonads Support Colonization, Survival, and Proliferation of Intraperitoneally Transplanted Xenogenic Germ Cells1

The production of xenogenic gametes from large-bodied, commercially important marine fish species in closely related smaller host fish species with short generation times may enable rapid and simple seed production of the target species. As a first step toward this goal, we assessed the suitability of chub mackerel, Scomber japonicus, as a small-bodied recipient species for xenogenic spermatogonial transplantation. Histological observation of the early gonadal development of chub mackerel larvae and transplantation of fluorescent-labeled spermatogonia from Nibe croaker, Nibea mitsukurii, revealed that 5.3-mm chub mackerel larvae were suitable recipients for successful transplantation. Intraperitoneally transplanted xenogenic spermatogonia efficiently colonized the gonads of these recipient larvae, and donor-derived Nibe croaker germ cells proliferated rapidly soon after colonization. Moreover, gonadal soma-derived growth factor (gsdf) mRNA, a gonadal somatic cell marker, was expressed in recipient-derived cells surrounding the incorporated donor-derived germ cells, suggesting that donor-derived germ cells had settled at an appropriate location in the recipient gonad. Our data show that xenogenic spermatogonial transplantation was successful in chub mackerel and that the somatic microenvironment of the chub mackerel gonad can support the colonization, survival, and proliferation of intraperitoneally transplanted xenogenic germ cells derived from a donor species of a different taxonomic family.

Development of Spermatogonial Cell Transplantation in Nibe Croaker, Nibea mitsukurii (Perciformes, Sciaenidae)

In a recently established system for intraperitoneal spermatogonial cell transplantation in salmonids, donor type A spermatogonia (type A SG) were microinjected into the peritoneal cavity of newly hatched larvae. Compared with salmonids, the larvae of marine teleosts are small and vulnerable to physiological and physical stresses, making it difficult to use them for cell manipulation. Herein, we developed type A SG cell transplantation in Nibe croaker (Nibea mitsukurii) by optimizing 1) the developmental stage of the donor testes used to prepare type A SG-enriched cell suspensions and 2) the timing and location of intraperitoneal cell transplantations to recipient larvae. Donor cells labeled with PKH26 fluorescent dye were transplanted into the peritoneal cavity of 3-, 4-, 5-, and 6-mm larvae using glass micropipettes. Consequently, 20.6% of the 4-mm larvae recipients survived for 3 wk, and 36.3% of the survivors had donor-derived cells in their gonads. The incorporated donor cells were identified as germ cells by germ cell-specific nuclear morphology and expression of a germ cell marker. In contrast, no donor type A SG were incorporated into the gonads of 6-mm recipient larvae. These data indicate that there is a distinct narrow window in the developmental stages of recipient larvae when exogenous type A SG can be incorporated into the gonads. The establishment of this system in pelagic egg-spawning marine teleosts would allow the creation of a new broodstock system in which a target species with a large body size and long generation time could be produced from related species with a small body size and short generation time.

Gonadotropin-dependent oocyte maturational competence requires activation of the protein kinase A pathway and synthesis of RNA and protein in ovarian follicles of Nibe, Nibea mitsukurii (Teleostei, Sciaenidae)

Luteinizing hormone- (LH)-dependent ovarian follicle maturation has been recently described in two stages for teleost fishes. The oocyte's ability to respond to the steroidal maturation-inducing hormone (MIH), also known as oocyte maturational competence (OMC), is acquired during the first stage+ADs- whereas the MIH-dependent resumption of meiosis occurs during the second stage. However, studies directly addressing OMC have been performed with a limited number of species and therefore the general relevance of the two-stage model and its mechanisms remain uncertain. In this study, we examined the hormonal regulation of OMC and its basic transduction mechanisms in ovarian follicles of the sciaenid teleost, Nibe (Nibea mitsukurii). Exposure to MIH +AFs-17,20+ACY-bgr+ADs--dihydroxy-4-pregnen-3-one or 17,20+ACY-bgr+ADs-,21-trihydroxy-4-pregnen-3-one] stimulated germinal vesicle breakdown (index of meiotic resumption) in full-grown follicles primed with human chorionic gonadotropin (HCG, an LH-like gonadotropin) but not in those pre-cultured in plain incubation medium. The induction of OMC by HCG was mimicked by protein kinase A (PKA) activators (forskolin and dibutyryl cyclic AMP), and blocked by specific inhibitors of PKA (H89 and H8) as well as inhibitors of RNA (actinomycin D) and protein (cycloheximide) synthesis. Forskolin-induced OMC was also inhibited by actinomycin D and cycloheximide. A strong activator of protein kinase C, PMA, inhibited HCG-dependent OMC. In conclusion, OMC in Nibe ovarian follicles is gonadotropin-dependent and requires activation of the PKA pathway followed by gene transcription and translation events. These observations are consistent with the two-stage model of ovarian follicle maturation proposed for other teleosts, and suggest that Nibe can be used as new model species for mechanistic studies of ovarian follicle differentiation and maturation in fishes.

Mechanical Behavior and Cross Linkages of Heat-induced Myosin Gel.

The tensile breaking properties of fish myosin gel were examined together with the investigation of the kinds of cross linkages of the heat-induced myosin gel. Sample myosin was extracted from croaker Nibea mitsukurii and was ground with 0.6M KCl. It was then heated to gel at 80°C for 20 min. The breaking strain along with the breaking stress were measured in concentrations ranging from 70 to 120mg myosin/g gel. The former was a maximum at around 85mg/g gel while the latter increased with a rise in myosin content. The breaking strain of myosin gel to which urea and guanidine hydrochloride was separately added decreased with the concentration of any reagents. Similar results were observed when sodium dodecylsulfate, a kind of surfactant, was added. On the contrary, in the case of the addition of 2-mercaptoethanol as a reducing reagent of the disulfide bond, the breaking strain was almost the same as that of the control. From the set of results, the hydrogen bond along with hydrophobic bond were found to play principal roles in the gellation of the myosin, while the disulfide bond contribution was negligible.

A possible correlation between environmental chemicals and pigment cell neoplasia in fish

A croaker (Nibea mitsukurii) has a high incidence of the skin melanoma, chromatophoroma, in a Pacific coastal area in Japan. A sea catfish (Plotosus anguillaris) bearing skin melanosis is also found in the same area. For elucidation of a correlation between these pigment cell neoplasms of the skin and environmental contaminants, an epidemiological survey was conducted to determine the distribution and prevalence of tumor-bearing fish. Based upon observations of a high prevalence of skin neoplasms near the discharge point for kraft pulp mills, experiments were conducted to determine the neoplastic induction efficiency of the effluent on the croaker and sea catfish species. Isolation and identification of mutagens in effluent extracts were carried out using the Ames test, followed by mass spectral analysis of mutagenic fractions. The effluent induced a chromatophoroma on one croaker of the 100 tested, and it induced pigment cell hyperplasia on 70 to 100% of the sea catfish. These skin neoplasms were grossly similar to those observed in the field. Five chloroacetones were identified from the Ames-positive fractions of the effluent, and tetrachlorocyclopentene-1,3-dione and two α-dicarbonyl compounds were also detected as mutagens. The above experiments indicate that the mutagenic contaminants found in kraft mill effluent may play an important role in the induction of skin neoplastic disease in fish.

Environment: peculiar pigment cell neoplasm in fish.

Chromatophoroma in the croaker (Nibea mitsukurii) showed a unique geographic distribution. The contribution of environmental chemicals to the cause of chromatophoroma in the feral croaker is considered likely on the basis of the following results in our studies. 1) Chromatophoroma was induced in tank-reared N. mitsukurii by administration of certain kinds of known carcinogens such as 7,12-dimethyl-benz(a)anthracene, N-methyl-N'-nitro-N-nitrosoguanidine, and nifurpirinol. 2) Local accumulation of pigment-cell hyperplasia in the catfish (Protosus anguillaris) showed similar tendencies to those of chromatophoroma in N. mitsukurii. 3) Removal of contaminated sediment from the harbor and the river appeared to reduce the incidence from 47% in 1973-1983 to about 20% in 1985-1987. 4) Waste water from a factory located at the station where the incidence of the neoplasm was the highest contained mutagenic substances such as chloroacetones and glyoxals [5]. Exposure of catfish to the waste water induced pigment-cell hyperplasia on the skin.

Environment: Peculiar Pigment Cell Neoplasm in Fish

Chromatophoroma in the croaker (Nibea mitsukurii) showed a unique geographic distribution. The contribution of environmental chemicals to the cause of chromatophoroma in the feral croaker is considered likely on the basis of the following results in our studies. 1) Chromatophoroma was induced in tank-reared N. mitsukurii by administration of certain kinds of known carcinogens such as 7,12-dimethyl-benz(a)anthracene, N-methyl-N'-nitro-N-nitrosoguanidine, and nifurpirinol. 2) Local accumulation of pigment-cell hyperplasia in the catfish (Protosus anguillaris) showed similar tendencies to those of chromatophoroma in N. mitsukurii. 3) Removal of contaminated sediment from the harbor and the river appeared to reduce the incidence from 47% in 1973-1983 to about 20% in 1985-1987. 4) Waste water from a factory located at the station where the incidence of the neoplasm was the highest contained mutagenic substances such as chloroacetones and glyoxals [5]. Exposure of catfish to the waste water induced pigment-cell hyperplasia on the skin.