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Artificial fertilization of cultured fish is essential for seed production using breeding techniques. However, in tuna species, the success rate of artificial fertilization is tremendously low. In this study, it was reported that the adequate procedure for ovulated egg collection and storage for artificial fertilization in kawakawa Euthynnus affinis. The collection of ovulated eggs was attempted using new techniques that disrupt only spawning activity without discontinuing ovulation. The available time to use ovulated eggs was also examined by assessing the optimal preservation process and temperature. As a result, artificial fertilization was effectively executed by assessing spawning time and thoroughly extracting ovulated eggs immediately after ovulation, with a success rate of 70% and an ovulation rate of 51.7%. Ovulated eggs could be stored with small quantities of ovarian fluid to sustain fertility. However, fertility was better preserved with Hanks' solution. Ovulated eggs with high productivity were achieved 3 h after egg extraction when maintained in Hanks' solution at 20 °C, leading to a supply of one-cell stage embryo for microinjection treatment constantly by continuously executing artificial fertilization. This systematic procedure permitted selective breeding by 1:1 mating between top-quality parental fish and applying several developmental engineering techniques to kawakawa breeding. [ABSTRACT FROM AUTHOR]

The horizontal stray field of the pulse septum is reduced at NewSUBARU by using an air-cored correction coil. This is designed and adjusted based on measurements using the stored electron beam.

Abstract To elucidate the molecular mechanisms underling hydration during oocyte maturation, we characterized the structure of Japanese eel (Anguilla japonica) novel-water selective aquaporin 1 (AQP1b) that thought to be involved in oocyte hydration. The aqp1b cDNA encodes a 263 amino acid protein that includes the six potential transmembrane domains and two Asn-Pro-Ala motifs. Reverse transcription-polymerase chain reaction showed transcription of Japanese eel aqp1b in ovary and testis but not in the other tissues. In situ hybridization studies with the eel aqp1b cRNA probe revealed intense eel aqp1b signal in the oocytes at the perinucleolus stage and the signals became faint during the process of oocyte development. Light microscopic immunocytochemical analysis of ovary revealed that the Japanese eel AQP1b was expressed in the cytoplasm around the yolk globules which were located in the peripheral region of oocytes during the primary yolk globule stage; thereafter, the immunoreactivity was observed throughout the cytoplasm of oocyte as vitellogenesis progressed. The immunoreactivity became localized around the large membrane-limited yolk masses which were formed by the fusion of yolk globules during the oocyte maturation phase. These results together indicate that AQP1b, which is synthesized in the oocyte during the process of oocyte growth, is essential for mediating water uptake into eel oocytes.

Abstract: Most behavioral studies on hermaphroditic fishes have focused on small‐sized species, which are tractable for research. Although many species of large hermaphroditic fishes are important fishery resources, their proximate mechanisms (visual, chemical and/or behavioral cues) in the social regulation of sex change have not been determined. Determination of these would inform resource management and aquaculture. In order to get closer to understanding the proximate mechanisms underlying the social regulation of female‐to‐male sex change in large hermaphroditic fishes, this study reports situations that induced female‐to‐male sex change in black‐spot tuskfish, Choerodon schoenleinii, a species of large protogynous fish, in massive laboratory tanks. The situations differed in the possibility of male‐to‐female tactile contact and in the group sex ratio, enabling us to infer plausible proximate mechanisms underlying sex change induction. Tactile contact between individuals is suspected to be closely related to the incidence of female‐to‐male sex change in C. schoenleinii. Visual and chemical cues alone may be insufficient to inhibit such sex changes. Male‐to‐female tactile contact may have an important influence on female‐to‐male sex change, i.e., inhibition of this sex change, in this species. The effect of sex ratio of a social group on the incidence of sex change may be due to the relative frequency and intensity of male tactile contact with each female, which may vary with the number of females. In the absence of a dominant male, tactile contact among females may affect the incidence of sex change, as well as determine which individuals change sex. [ABSTRACT FROM AUTHOR]

To elucidate the molecular mechanisms underling hydration during oocyte maturation, we characterized the structure of Japanese eel (Anguilla japonica) novel-water selective aquaporin 1 (AQP1b) that thought to be involved in oocyte hydration. The aqp1b cDNA encodes a 263 amino acid protein that includes the six potential transmembrane domains and two Asn-Pro-Ala motifs. Reverse transcription-polymerase chain reaction showed transcription of Japanese eel aqp1b in ovary and testis but not in the other tissues. In situ hybridization studies with the eel aqp1b cRNA probe revealed intense eel aqp1b signal in the oocytes at the perinucleolus stage and the signals became faint during the process of oocyte development. Light microscopic immunocytochemical analysis of ovary revealed that the Japanese eel AQP1b was expressed in the cytoplasm around the yolk globules which were located in the peripheral region of oocytes during the primary yolk globule stage; thereafter, the immunoreactivity was observed throughout the cytoplasm of oocyte as vitellogenesis progressed. The immunoreactivity became localized around the large membrane-limited yolk masses which were formed by the fusion of yolk globules during the oocyte maturation phase. These results together indicate that AQP1b, which is synthesized in the oocyte during the process of oocyte growth, is essential for mediating water uptake into eel oocytes. [ABSTRACT FROM AUTHOR]

Transcription activator-like effector (TALE) nucleases (TALENs) mediated gene editing methods are becoming popular and have revealed the staggering complexity of genome control during development. Here, we present a simple and efficient gene knockout using TALENs in kawakawa, Euthynnus affinis, using slc24a5. We examined slc24a5 gene expression and functional differences between two TALENs that hold the TALE scaffolds, +153/+47 and +136/+63 and target slc24a5. Developmental changes in slc24a5 transcripts were seen in early-stage embryos by real-time PCR; slc24a5 expression was first detected 48 h post fertilization (hpf), which increased dramatically at 72 hpf. Four TALENs, 47- and 63-type of two different target loci (A and B), respectively, were constructed using Platinum TALEN and evaluated in vitro by a single-strand annealing (SSA) assay. TALEN activities were further evaluated in vivo by injecting TALEN mRNAs in the two-cell stage of the zygote. Most of the TALEN-induced mutants showed mosaic patterns in the retinal pigment epithelium (RPE) and fewer melanin pigments on the body at 72 hpf and later when compared to the control, implying the gene's association with melanin pigment formation. A heteroduplex mobility assay (HMA) and the genome sequence further confirmed the TALEN-induced mutations of substitution, insertion, and deletion at an endogenous locus. [ABSTRACT FROM AUTHOR]

The fish egg is surrounded by a thick envelope called the chorion. The fertilizing spermatozoon enters the egg through a canal-like structure in the chorion, the micropyle. Examination of micropyle at fertilization is difficult if eggs are large and have no distinct landmarks surrounding the micropyle, or if they are positively buoyant in water. Eggs of many commercially important fishes (e.g., flounder, sea bream and eel) are buoyant in water or only slightly adhere to solid objects (e.g., sands, rock and water plants), which makes observation of spermatozoa at fertilization difficult. Here, we report that such eggs can be firmly attached to plastic and glass dishes that have been previously coated with poly-L-lysine. These adhering eggs can be fertilized and develop normally on the dishes. Observations of micropyles of three fish species, before and after sperm entry are presented. [ABSTRACT FROM AUTHOR]

Sperm of the Pacific herring are immotile at spawning. Two egg-derived molecules are capable of initiating sperm motility. One is herring sperm activating protein(s) (HSAPs) and the other is sperm motility initiation factor (SMIF). These two motility initiators differ in their location and association with the chorion, and in thier isoelectric points and molecular weights. In this study we have investigated the roles of these two inducers with respect to motility and fertilization. Using computer analysis of sperm motility, we found that HSAPs, as well as the Cterminal HSAPs peptide, elicit a linear motility pattern, while SMIF induced a highly circular and asymmetric pattern. HSAPs induced a two-fold increase in intracellular calcium, whereas SMIF induced a four-fold increase of motility initiation. SMIF-exposed sperm, preloaded with BAPTAAM, showed a more linear motility and this motility trajectory decreased with their fertilizing capability. The difference in intracellular calcium levels between HSAPs and SMIF is consistent with the observed linear and circular motility. In the absence of SMIF, HSAPs do not support fertilization. Fertilization is rescued in these experiments if SMIF is reintroduced. We propose that diffusible HSAPs are not essential for fertilization, but enhance sperm-egg collisions via linear motility. SMIF, which is bound to the micropylar region of the chorion, is required for fertilization and induces circular motility that is a prerequisite for sperm to enter the micropylar canal and fertilize the egg. [ABSTRACT FROM AUTHOR]

Surveys of the estrogenic activity in coastal areas of Japan were performed by using two serum vitellogenins (Vg-530 and Vg-320) of male Japanese common goby Acanthogobius flavimanus as biomarkers. To evaluate the relationship between the Vg concentrations in the serum of male fish and the concentrations of environmental estrogens in seawater, 3 week exposure experiments were performed using estradiol (E2), nonylphenol (NP) and bisphenol A (BPA). From the results of the E2 exposure, mean concentrations of the Vg and the ratio of Vg-positive fish increased from actual concentration at 10.5 ng/L E2 treatment. Exposure of NP and BPA showed that the 19.0 µg/L NP treatment induced the Vg but 133.7 µg/L BPA did not. As for the survey of the estrogenic activity in coastal areas, the mean Vg concentrations and ratio of Vg-positive fish in some urban areas were similar to values of the 10.5 ng/L E2 treatment. Although E2 and NP were detected in the seawater from urban areas, the concentrations were concluded as not sufficient to independently induce the Vg. Considering these results, it is concluded that the total estrogenic activity in some urban areas is dependent not only on E2 but synergistically with other environmental estrogens. [ABSTRACT FROM AUTHOR]

Annual changes in testicular development and serum levels of androgen and progestins were investigated using captive male spotted halibut Verasper variegatus in order to obtain basic information on the reproductive physiology of this species. Based on the histological observation of testes, male maturity was divided into the following five stages: spermatogonial proliferation stage (July to September), early spermatogenic stage (October), late spermatogenic stage (November to December), functional maturation stage (January to March) and recovery stage (May). Spermatogenesis rapidly progresses during the autumn from October and December and continues until the functional maturation stage (spawning period of female). Serum levels of 11-ketotestosterone gradually increased from August and rapidly increased to a peak (2.8 ± 0.5 ng/mL) in November, suggesting that this steroid is involved in spermatogenesis in this species. Serum 17,20β-dihydroxy-4-pregnen-3-one levels showed a single small peak (0.55 ± 0.05 ng/mL) in November, suggesting the possibility of the involvement of this steroid hormone in the late spermatogenic period. [ABSTRACT FROM AUTHOR]

Because blood vitellogenin (Vg) has been considered a biomarker for environmental estrogens, the basal levels of Vg and 17β-estradiol (E2 ) were determined in male Japanese whiting reared under natural conditions. Serum levels of Vg and E2 were measured and gonadal development was assessed by gonadosomatic index (GSI) and histological observation in 8–10 male fish at monthly intervals throughout the annual reproductive cycle. Serum E2 was <60 pg/mL throughout the study period. In contrast, serum Vg exhibited seasonal changes: serum levels of Vg gradually increased from April to May (mean 63 ± 13 ng/mL and 124 ± 48 ng/mL in April and May, respectively), and then reached a peak value (mean 352 ± 68 ng/mL) in June. Thereafter, serum Vg gradually decreased, reaching undetectable levels (<50 ng/mL) in October. Serum levels of Vg tended to increase in the male fish in which the GSI was >1%. Histological observation revealed that testes in such male fish were in active spermatogenesis and then all of the testes of male fish in which serum Vg decreased to ND levels were regressed. These results suggest that Vg productive potency (sensitivity to estrogens) may increase in the spermatogenic stage, resulting in production of Vg in response to very low levels of natural or xenobiotic estrogens. [ABSTRACT FROM AUTHOR]

ABSTRACT: The present study investigates the relationship between oocyte development and serum steroid hormone levels in captive Pacific herring, Clupea pallasii , during the first reproductive cycle. The process of oocyte development in Pacific herring belongs to the group-synchronous type. Maturity of the ovary was divided into six periods based on histological observation (i.e. immature (April to September), onset of vitellogenesis (August to October), progress of vitellogenesis (October to December), completion of vitellogenesis (December to March), maturation and spawning (March to April) and spent (late April)). The pattern of seasonal change in the gonadosomatic index (GSI) well reflected the ovarian maturity. Serum vitellogenin levels showed good correlation with change in GSI, which increased from September to a peak (4.2 ± 0.3 mg/mL) in March. Serum estradiol-17β (E2) levels elevated from September and reached a peak (15.8 ± 4.2 ng/mL) in December, and remained comparatively high until March, suggesting that the active vitellogenin synthesis during vitellogenesis is controlled by the high E2 level. 17,20β-Dihydroxy-4-pregnen-3-one showed a single sharp peak (2.4 ± 0.28 ng/mL) in early April of the second year, suggesting it was a maturation-inducing steroid in this species. [ABSTRACT FROM AUTHOR]

ABSTRACT: The present study investigates the relationship between testicular development and serum steroid hormone levels in captive Pacific herring Clupea pallasii during the first reproductive cycle. The maturity of the testis was divided into five periods based on histological observation. These are early spermatogenic stage (April to July), mid-spermatogenic stage (August to November), late spermatogenic stage (December to March), functional maturation stage (early April) and spent stage (late April). The pattern of seasonal change in gonadosomatic index (GSI) clearly reflected testicular maturity. 11-Ketotestosterone (11-KT) levels increased from October to a peak level (6.58 ± 1.87 ng/mL) in January, and were maintained at this level until March. In contrast, testosterone levels were consistently low, less than 1 ng/mL, at all times. These results suggest that 11-KT is the predominant androgen that controls spermatogenesis in this species. 17,20β-Dihydroxy-4-pregnen-3-one (DHP) showed a single sharp peak (3.38 ± 0.35 ng/mL) in early April of the second year, suggesting that milt production is induced by DHP as in some other teleost species. [ABSTRACT FROM AUTHOR]

ABSTRACT During spermatogenesis, many proteins are synthesized in the testis prior to the completion of sperm maturation. Many components are involved in the testicular protein synthesis and one of the components that is implicated in the polypeptide chain elongation is elongation factor 1α. In the present study, the molecular cloning of elongation factor 1α (EF-1α) was conducted from a testis cDNA library of the Nile tilapia. The cDNA for tilapia EF-1α (tEF-1α) contains a complete open reading frame encoding 462 amino acids. The predicted amino acid sequence of EF-1α shows an approximate 90% similarity to those identified in other teleost fish, such as medaka, sea bream and zebrafish. Northern blotting revealed that the gene is expressed in all of the examined tissues and in ovulated eggs. The results of in situ hybridization indicate that the gene is expressed specifically in Leydig cells in testis, suggesting the involvement of EF-1α as an actin-binding protein in the cluster formation of Leydig cells. In the ovary, the gene is expressed in the perinucleolus stage of oocytes, suggesting that EF-1α is also implicated in oocyte growth. [ABSTRACT FROM AUTHOR]

Sperm that have acquired potential for motility are kept immotile in seminal plasma in the teleost, Nile tilapia. In order to investigate the mechanism of immobilization, several experiments were performed using a previously characterized monoclonal antibody (TAT-30) against a molecular weight (Mr) = 120 000 protein that is secreted by Sertoli cells and epithelial cells of the sperm duct, and is also bound to the head of the spermatozoon. First, we assessed sperm motility in the seminal plasma protein fraction (SPP), and demonstrated that the sperm motility is inhibited by SPP in a concentration-dependent manner. Furthermore, sperm motility was recovered if SPP was pretreated with TAT-30, suggesting that the TAT-30 antigen is one of the components of the sperm immobilizing factor. Calibration by gel filtration followed by sodium dodecylsulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting with TAT-30 demonstrated that the sperm immobilizing factor was more than Mr = 1 000 000 in seminal plasma, suggesting that it is a homopolymer of the Mr = 120 000 – TAT-30 positive protein. Additionally, lectin blot analysis showed that the TAT-30 antigen was reactive with Lens culinarin agglutinin (LCA) and Conavalia ensiformis agglutinin (ConA), indicating that it is a glycoprotein. Immunohistochemical studies showed that the TAT-30 antigen was localized specifically on the heads of spermatozoa and on the apical surface, lysosomes and rough endoplasmic reticulum of Sertoli cells. [ABSTRACT FROM AUTHOR]

Two types of spiggin cDNAs were isolated from the three-spined stickleback. Northern blot analysis revealed that glue protein is composed of multiple spiggin molecules, and that their synthesis in kidney is strongly up-regulated by androgens. [ABSTRACT FROM AUTHOR]

We investigated the effects of endocrine-disrupting chemicals (EDCs) on the expression of ubiquitin C-terminal hydrolase (UCH) mRNA in the testis and brain of the Japanese common goby, Acanthogobius flavimanus. The cDNA sequence of goby UCH contained an open reading frame encoding 220 amino acid residues (Mr=24,223) with 51.3% overall sequence identity with human and mouse UCHL1. A competitive PCR assay was used to quantify the levels of UCH mRNA in the testis and brain of male gobies after exposure to bisphenol A, nonylphenol, or estradiol-17β for 3 weeks. Exposure to estradiol-17β at a nominal concentration of 100ng/L induced significant increase in UCH mRNA levels in both testis and brain (P<0.05), whereas exposure to nonylphenol induced a significant decrease in UCH mRNA levels in the testis (P<0.01). These results suggest that EDCs can either positively or negatively regulate UCH mRNA levels. [Copyright &y& Elsevier]