Hirohiko Kagawa

Artificial induction of maturation and fertilization in the Japanese eel, Anguilla japonica.

Repeated injections of salmon pituitary extract (20 mg per fish per week) induced vitellogenesis in feminized, cultivated Japanese eels (Anguilla japonica). Oocytes were attained at the migratory nucleus stage after 11 or 12 injections. Addition of 17,20β-dihydroxy-4-pregnen-3-one (DHP) into the incubation medium induced germinal vesicle breakdown (GVBD) in the oocytes at the migratory nucleus stage. An injection of DHP (2 µg g-1 BW), given 24h after an injection of salmon pituitary extract (20 mg fish-1), succeeded in inducing maturation and ovulation in females which contained occytes at the migratory nucleus stage. Most fish ovulated 15–18h following the DHP injection. Eggs that were ovulated within 15h after the DHP injection showed high fertility and hatchability, but eggs ovulated 18 or 21h after the DHP injection, showed considerably lower fertility and hatchability. A delay between ovulation and stripping of the eggs rapidly decreased both the fertility and hatchability within 6–9h after ovulation, indicating that artificial fertilization must be carried out immediately after ovulation. Repeated injections of human chorionic gonadotropin (hCG) at a concentration of 1 IU g-1 BW week-1 induced spermatogenesis, spermiation, and the acquisition of potential for sperm motility in cultivated males. Most males spermiated after the fifth or sixth injection of hCG, and the milt weight gradually increased and remained constant (1–2 g) from the 11th to 31th injection. Sperm motility peaked 24h after each weekly injection, and decreased from the 3rd day after the injection. Potassium ions are an essential constituent for the maintenance of motility in the eel spermatozoa. Artificial seminal plasma containing 15.2 mM KCl is applicable as a milt diluent. Using these techniques developed for female and male eels, we have succeeded in obtaining many fertilized eggs from cultivated eels.

Changes in fertilization and hatching rates with time after ovulation induced by 17, 20β-dihydroxy-4-pregnen-3-one in the Japanese eel, Anguilla japonica

Abstract This study examined the time to ovulation in artificially matured Japanese eel following an injection of 17, 20β-dihydroxy-4-pregnen-3-one (DHP), and changes in fertilization and hatching rates at various times after ovulation. Thirteen females, showing body weight indices (body weight/initial body weight × 100) of 119 ± 2.3% with oocytes at the migratory nucleus stage after 9–12 weekly injections of salmon pituitary extract (20 mg pituitary powder /fish/week), received an injection of salmon pituitary extract (20 mg/fish) followed 24 h later by a DHP injection (2μg/g BW) for induction of ovulation. Eleven of 13 females ovulated within 23 h of the final injection; two, seven, and two females ovulated at 14–17 h, 17–20 h and 20–23 h after the final injection, respectively. The remaining two did not ovulate within 26 h of the injection. Eggs from three out of the 11 ovulated females showed relatively high percent fertility (89.6-39.5%) and hatching rates (47.6-15.3%) when they were fertilized after the first detection of ovulation. Both percent fertilization and hatching rates of eggs retained in the body cavity decreased rapidly within 6–9 h after ovulation. These results indicate that ovulation induced by DHP treatment occurs after a short but well-defined period of time, and that artificial fertilization must be carried out immediately after ovulation in order to obtain good quality eggs.

Production of leptocephali of Japanese eel (Anguilla japonica) in captivity

Despite intensive research on wild and captive eels, no resource has so far provided access to all life cycle stages of the Japanese eel Anguilla japonica. The transition from the preleptocephalus (newly hatched larva) to the leptocephalus stage (typical leaf-like eel larva) has, therefore, remained the missing link in the eel life cycle. We recently found that a slurry-type diet made from shark egg powder is suitable feed for captive-bred eel larvae. The larvae were successfully reared with this diet in aquaria for 100 days and raised to 22.8 mm in total length (TL). Age, TL, and body proportions of the reared specimens overlapped with those of wild leptocephali. We revealed for the first time the transition from the preleptocephalus to the leptocephalus stage of the eel.

Unique expression of gonadotropin-I and -II subunit genes in male and female red seabream (Pagrus major) during sexual maturation.

Abstract Two distinct gonadotropins (GTHs) have been demonstrated in a number of teleost fishes. Although the physiological roles of GTHs have been extensively studied in salmonids, little is known about their biological functions in nonsalmonid fishes. In this study, to elucidate the role of GTH-I and GTH-II in reproduction, we cloned the α-glycoprotein subunit (αGSU) and gonadotropin β subunits (Iβ and IIβ) of red seabream using the 5′- and 3′-RACE methods and used these cDNA probes to reveal changes in mRNA levels of each subunit during sexual maturation of both male and female red seabream. The nucleotide sequences of αGSU, Iβ, and IIβ are 629, 531, and 557 base pairs long, encoding peptides of 117, 120, and 146 amino acids, respectively. The deduced amino acid sequence of each mature subunit showed high homology with those of other teleosts. Northern blot analysis showed that Iβ mRNA levels of males increase in association with gonadal development, whereas those of females remain low throughout sexual maturation, indicating sexual dimorphism in the expression pattern of Iβ. In contrast, IIβ mRNA levels of both sexes are maintained at high levels from the beginning of gametogenesis to spawning season. These results are different than those of salmonids and suggest that GTH-I may have important roles in male, but not female, gametogenesis. GTH-II may be involved in regulation of early and late gametogenesis in both male and female red seabream.

Effects of Luteinizing Hormone and Follicle-Stimulating Hormone and Insulin-Like Growth Factor-I on Aromatase Activity and P450 Aromatase Gene Expression in the Ovarian Follicles of Red Seabream, Pagrus major

Abstract To clarify the mechanism of estradiol-17β production in the ovarian follicle of red seabream, in vitro effects of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and insulin-like growth factor (IGF-I) on aromatase activity (conversion of testosterone to estradiol-17β) and cytochrome P450 aromatase (P450arom) mRNA expression in ovarian fragments of red seabream were investigated. Of the growth factors used in the present study, only IGF-I stimulated both aromatase activity and P450arom gene expression in the ovarian fragments of red seabream. LH from red seabream pituitary, but not FSH, stimulated both aromatase activity and P450arom gene expression. IGF-I slightly enhanced the LH-induced aromatase activity and P450arom gene expression. These data and our previous results indicate that LH, but not FSH, stimulates estradiol-17β production in the ovarian follicle of red seabream through stimulation of aromatase activity and P450arom gene expression and IGF-I enhances the LH-stimulated P450arom gene expression.

Purification of gonadotropins (PmGTH I and II) from red seabream (Pagrus major) and development of a homologous radioimmunoassay for PmGTH II

Two gonadotropic glycoproteins (PmGTH I and II) were purified by ion-exchange chromatography, gel filtration and preparative SDS-PAGE, from pituitaries of red seabream, a marine teleost which has an asynchronous-type ovary and spawns almost daily during the spawning season. The glycoproteins were composed of distinct subunits and the molecular weights were estimated to be 32 and 38 kDa for PmGTH I and PmGTH II, respectively. Both PmGTH I and II were active in two homologous bioassays: in vitro oocyte maturation and/or in vitro estradiol-17β production assays. These two GTHs were distinct in electrostatic properties, molecular weight, stability and yields from pituitaries during the spawning season. These properties suggest that PmGTH I and II correspond to salmon GTH I and II, respectively.A homologous radioimmunoassay with which to measure PmGTH II was developed using a rabbit antiserum against the β subunit of PmGTH II and intact PmGTH II as standards and radioactive competitors. Competition curves for red seabream plasma and pituitary extract were parallel to the standard curve, while PmGTH I had low cross-reactivity (3.1 %) with the antibody. This specific RIA system showed an in vivo LHRHa induced GTH surge in the plasma of female red seabream.

Effects of gonadotropin-releasing hormone agonist and dopamine antagonist on hypothalamus–pituitary–gonadal axis of pre-pubertal female red seabream (Pagrus major)

The effects of GnRH agonist (GnRHa) on the hypothalamus-pituitary-gonadal axis were studied in female pre-pubertal red seabream. Sexually immature 16-month-old fish were implanted intramuscularly with cholesterol pellets containing GnRHa or GnRHa in combination with domperidone, putative dopamine antagonist, and reared for 10-20 days. In both GnRHa and GnRHa+domperidone implanted groups, vitellogenesis was observed on Day 10 and ovulation was observed on Day 20, while ovarian development was not observed in the control fish throughout the experimental period. The levels of GnRH receptor mRNA were significantly higher in both GnRHa implanted groups than in the control. The expressions of all three gonadotropin subunit genes were up-regulated and serum luteinizing hormone levels were increased by the GnRHa implantation. Serum testosterone and estradiol-17beta levels were also increased on Day 10 and maintained high levels on Day 20. On the other hand, seabream (sb) GnRH mRNA levels in the brain were relatively low and unchanged in all experiment groups. The present study first shows that GnRH alone can induce precocious puberty in red seabream. These results indicate that the system of pituitary-gonadal axis has already been developed in 16-month-old fish and the commencement of sbGnRH secretion may be an important physiological event for the onset of puberty in the red seabream.

Immunocytochemistry on the Localization of 5-Hydroxytryptamine in Monkey and Rabbit Taste Buds

Immuno-electron microscopy with the protein A-gold method demonstrated immunoreactive gold particles against 5-hydroxytryptamine localized in cored vesicles aggregating around presynaptic terminals of the gustatory cells in monkey and rabbit taste buds. The positive reactions were also found in the intragemmal and subepithelial nerve fibers. The role of these cored vesicles in taste transduction is still uncertain but the data strongly suggest that they may participate in a serotonergic modulation of a cholinergic synaptic transmission from the gustatory cells to the nerve endings.

Distinct expression of GnRH genes in the red seabream brain

This paper reports the molecular cloning of a cDNA encoding the precursor of seabream gonadotropin-releasing hormone (prepro-sbGnRH) and the localization of salmon GnRH (sGnRH) and seabream GnRH (sbGnRH) expressing neurons in the brain of the red seabream (Pagrus major). The cloned prepro-sbGnRH cDNA has a 285 bps open reading frame encoding a 23 amino acid signal peptide, a 10 amino acid sbGnRH, the cleavage site (Gly-Lys-Arg), and a 59 amino acid GnRH-associated peptide. The expression of sGnRH and sbGnRH peptides, and prepro-sGnRH and prepro-sbGnRH mRNA were studied using immunocytochemistry and non-radioactive in situ hybridization, respectively. We found cell bodies that reacted positively with both the sGnRH cRNA probe and anti-sGnRH serum, but not with the sbGnRH cRNA probe or anti-sbGnRH serum in the ganglion of the terminal nerve. Cell bodies that reacted positively with the sbGnRH cRNA probe, anti-sbGnRH serum, and anti-sGnRH serum, but negatively with the sGnRH cRNA probe were found in the preoptic area (POA). Immunocytochemistry showed that a distinct bundle of axons arises in the POA which projected to the pituitary gland. These results suggest that sbGnRH is the most relevant hypophysiotropic form of GnRH.

Seasonal variation of the three native gonadotropin-releasing hormone messenger ribonucleic acids levels in the brain of female red seabream

We studied the seasonal variation of the expression of genes encoding the three native gonadotropin-releasing hormones (GnRHs), namely salmon(s) GnRH, chicken(c) GnRH-II, and seabream(sb) GnRH in red seabream, Pagrus (Chrysophrys) major, in order to better understand the regulatory mechanisms of GnRH gene expression by environmental and endocrine factors. Female red seabream, reared under natural conditions, were collected monthly or bimonthly from October to June, and the levels of the three distinct GnRH messenger ribonucleic acids (mRNAs) in the brains of those fish (n = 4-6) were determined by ribonuclease (RNase) protection analysis. The levels of sbGnRH mRNA correlated well with the observed ovarian histology; the levels of sbGnRH mRNA of immature fish in October and December were low, and increased in February and March in conjunction with active vitellogenesis. The sbGnRH mRNA levels reached a maximum level in April (spawning season), after which they rapidly decreased together with the observed ovarian regression in June. In contrast, the levels of sGnRH mRNA showed no variation, while those of cGnRH-II mRNA were elevated only slightly in March and April. The increase in sbGnRH mRNA levels correlates with the increase in day length, water temperature and serum steroids levels, suggesting that these factors are candidates for regulators of sbGnRH synthesis.