Isao Hanyu

Differences in salmon GnRH and chicken GnRH-II contents in discrete brain areas of male and female rainbow trout according to age and stage of maturity

We have developed sensitive and specific radioimmunoassays (RIA) for salmon gonadotropin-releasing hormone (sGnRH) and chicken GnRH-II (cGnRH-II). Synthetic sGnRH and cGnRH-II(2-10) were conjugated to bovine serum albumin and injected into rabbits to raise specific antisera. The antiserum against sGnRH showed cross-reactivities of 1.58 and 0.08% for cGnRH-II and lamprey GnRH, respectively. The antiserum against cGnRH-II showed cross-reactivities of 0.05 and 0.01% for sGnRH and lamprey GnRH, respectively. Both antisera were observed not to cross-react with mammalian GnRH and cGnRH-I or other peptide hormones. Synthetic sGnRH and cGnRH-II were iodinated using the chloramine-T method. The iodinated GnRH was purified by HPLC using a reverse-phase C18 column. The RIA system was developed as a double antibody method. Brain extracts of rainbow trout showed displacement curves which were parallel to the sGnRH and cGnRH-II standards in each RIA. HPLC analysis followed by RIA has revealed that rainbow trout brain contains two types of GnRH: sGnRH and cGnRH-II. Total sGnRH content in the brain was about three-fold higher than that of cGnRH-II. In the olfactory bulbs, telencephalon, optic tectum-thalamus, hypothalamus, and pituitary, sGnRH content (per region) was higher than cGnRH-II content, whereas cerebellum and medulla oblongata contained much more cGnRH-II than sGnRH. sGnRH content in the optic tectum-thalamus and pituitary was the highest in 1-year-old immature fish and 3-year-old mature fish, respectively. Medulla oblongata showed the highest cGnRH-II content in all groups. sGnRH concentrations (per milligram of protein) were high in the pituitary and intermediate in the olfactory bulbs, hypothalamus, and telencephalon. In all groups, the cGnRH-II concentration was high in the medulla oblongata, whereas the concentration in the olfactory bulbs and pituitary gland was below the detectable limit in most individuals.

Hormone changes during ovulation and effects of steroid hormones on plasma gonadotropin levels and ovulation in goldfish

Plasma hormone changes during ovulation and the effects of steroid hormones on plasma gonadotropin (GtH) levels and ovulation were studied in the female goldfish. Ovulation was induced by raising water temperature from 12 to 20 degrees. Plasma gonadotropin levels exhibited a gradual rise during the latter half of the light phase. This was then followed by a surge in GtH, showing a peak at the time of ovulation in the dark phase. After ovulation, GtH levels decreased rapidly by the next light phase. Plasma 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOH-P) and testosterone showed a peak before ovulation, and then decreased by the time of ovulation. Estradiol-17 beta (E2) showed moderate levels during the GtH surge. No marked decrease of E2 levels was observed before the commencement of the GtH surge. Administration of testosterone or E2 prior to elevating the water temperature did not inhibit the occurrence of the GtH surge and ovulation. Likewise, administration of neither 17 alpha-hydroxyprogesterone nor 17 alpha,20 beta-diOH-P affected plasma GtH levels at 20 degrees. The present study shows a clear picture of the hormone changes that occur during spontaneous ovulation in goldfish. The results of administering steroids prior to ovulation does not support the hypothesis that a decline in E2 levels triggers the GtH surge and ovulation.

Gonadotropin surge during spawning in male goldfish

Changes in plasma hormone levels during spawning were studied in male goldfish. Blood samples were analyzed for gonadotropin (GtH), testosterone, 11-ketotestosterone (11-KT), and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOH-P) in males, and for GtH in females. Spawning was induced by raising water temperature from 12 to 20 degrees in sexually mature goldfish kept in pairs. Plasma GtH levels in males showed a marked increase (GtH surge) which was synchronous with the preovulatory surge in females, and peaked at the onset of spawning. Plasma levels of testosterone and 17 alpha, 20 beta-diOH-P increased almost at the same time as the GtH surge in males, whereas 11-KT levels remained low during the spawning. The small increase in GtH in males could be induced in the absence of females by raising the water temperature, but the levels were significantly enhanced by the presence of ovulatory females. A large amount of milt was observed during spawning, and the amount was correlated with plasma levels of GtH. Increased levels of GtH may be involved in milt production. We can propose that the synchronous GtH surge in both sexes causes ovulation and milt production to occur at the same time, favoring a higher rate of fertilization of the eggs.

Hormone changes during the ovulatory cycle in goldfish

Hormone profiles during the ovulatory cycle were studied in goldfish. Blood samples were taken from female goldfish every 4 days between 1400 and 1700 hr during the course of repeated ovulations for a duration of 3 months, and plasma hormone levels of 3 days before and after ovulation were compared. Plasma gonadotropin (GtH) levels did not show significant changes except a surge for ovulation, but tended to show higher levels before the surge than those after the surge. Plasma testosterone before ovulation showed significantly higher levels compared with those after ovulation. Plasma estradiol-17 beta (E2) levels remained low for 3 days prior to ovulation. Postovulation E2 levels that were significantly higher than the preovulation levels were kept elevated and declined on the third day after ovulation. These results indicate that E2 is mainly produced in the first part of the ovulatory cycle and testosterone in the latter part followed by the GtH surge and ovulation at the end of the cycle. This shift in steroid pattern from E2 to testosterone seems to be similar to those observed in salmonid fishes except for the time scale. The synchrony of ovulation in goldfish is also discussed in relation to physiological and external factors which influence the occurrence of ovulation.

Daily cycles in plasma melatonin levels under long or short photoperiod in the common carp, Cyprinus carpio.

A radioimmunoassay (RIA) for plasma melatonin (MLT) was simplified for use with the common carp (Cyprinus carpio). Plasma was partially purified with Sep-Pak C18 cartridge before RIA. The inhibition curves for the Sep-Pak C18 fraction from the plasma of carp, goldfish (Carassius auratus), yellow tail (Seriola quinqueradiata), Japanese eel (Anguilla japonica), bora (Mugil cephalus cephalus), kisu (Sillago japonica), ishigarei (Kareius bicoloratus), and shimaisaki (Rhyncopelates oxyrhynchus) were parallel with the MLT standard curve. There was a highly significant correlation between MLT added to carp plasma and that which was recovered (r = 0.997, P less than 0.01). Intraassay coefficients of variation at low, medium, and high levels were 4.7, 5.2, and 6.4, respectively. Interassay coefficients of variation at low, medium, and high levels were 11.8, 8.2, and 24.1%, respectively. The lower limit of detection was 11 pg/tube. Plasma MLT levels were investigated every 2 hr in carp under 16L-8D and 8L-16D at 24 degrees. Under both photoperiods, MLT levels showed marked daily patterns, i.e., the levels were high (220-540 pg/ml) during the dark phase and low (23-104 pg/ml) during the light phase. Tricaine methanesulfonate markedly interfered with the MLT measurements, while ethylcarbamate did not show any significant influence. Sexual difference in plasma MLT levels in carp was not observed. These observations suggest that MLT is an important hormone in photoperiodism and/or a circadian rhythm in fish.

Circadian rhythms of melatonin secretion from superfused goldfish (Carassius auratus) pineal glands in vitro

A flow-through, whole-organ culture (superfusion) system was developed, and goldfish pineal glands were maintained at 25 degrees under light-dark (LD) 12:12 cycles, reversed LD 12:12 cycles, continuous dark (DD), or continuous light (LL) conditions for 48 hr. Under LD 12:12 and reversed LD 12:12 cycles, superfused pineal glands showed a rhythmic melatonin secretion: Scotophase was associated with high titers and photophase with low titers. The melatonin secretion rhythms persisted for two cycles under DD conditions, whereas nocturnal rises were suppressed under LL conditions. After the transition from LL to DD conditions on the third day, melatonin showed a nocturnal increase. These results indicate that melatonin secretion from the superfused goldfish pineal gland is directly photosensitive and that the goldfish pineal gland harbors a circadian oscillator which generates melatonin secretion rhythms.

Endocrine profiles during the short reproductive cycle of an autumn-spawning bitterling, Acheilognathus rhombea ☆

When autumn-spawning bitterling, Acheilognathus rhombea (known as the kanehira bitterling in Japan) were held under a constant short photoperiod (10 hr light-14 hr dark) at 25 degrees during their natural breeding season, the females spawned regularly at intervals of about 5 days. During each 5-day period, the oocytes passed through three distinct physiological stages: vitellogenesis (3 days), maturation (1 day), and spawning (1 day). During the maturational stage, germinal vesicle breakdown (GVBD) was first observed at 18 hr and was complete by 21 hr. Ovulation occurred at 24 hr. Steroid measurements made on plasmas collected from fish throughout their reproductive cycle showed that estradiol-17 beta levels were about 12 ng/ml at the beginning of the vitellogenic stage and about 5 ng/ml during the other stages. Testosterone levels were about 2 ng/ml during the vitellogenic stage, but fell to about 0.5 ng/ml during maturation and spawning. Progesterone levels were very low throughout the cycle and showed little variation. Both 17 alpha-hydroxyprogesterone and 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one levels were both very low (0.5 ng/ml) up until 12 hr on Day 4 (maturation), at which time they increased rapidly. The levels reached peaks of 4 and 7 ng/ml, respectively, at 18 hr and reached basal levels again at 8 hr the following day. GtH-like glycoprotein levels also began to increase at 12 hr on Day 4, but peaked later, at the time of ovulation (24 hr).

Effects of sex steroids on the smoltification of masu salmon, Oncorhynchus masou

Testosterone (T; 25-micrograms/g diet), estradiol-17 beta (E2; 2.5-, 25-micrograms/g diet), and 5 alpha-dihydrotestosterone (DHT; 2.5-, 25-micrograms/g diet) were administered to yearling masu salmon from February 3 through May 8. T and E2 inhibited the natural smoltification in spring and stimulated gonadotropin (GtH) accumulation in the pituitary, but DHT had no effect. T (25-micrograms/g diet) was administered to underyearling masu salmon from September 30 through December 14. At the end of the T treatment, fish body color darkened and pituitary GtH content increased in the T-treated group. On May 8 in the next spring, however, plasma T levels and pituitary GtH contents already decreased and smoltification occurred in both control and T-treated groups. On October 29, underyearling masu salmon reared under the short daylength (8 L:16 D) for 4 months were switched to a long daylength (16 L:8 D) and simultaneously administered with T (25-micrograms/g diet) or 11-ketotestosterone (11-KT; 25-micrograms/g diet). On December 17, the proportion of smolts was 52.8% in the control, whereas T and 11-KT administration inhibited smoltification under the artificially increased daylength. Pituitary GtH content significantly increased both in the T-treated and 11-KT-treated groups, though it was much lower in the latter. These results indicate that the springtime smoltification which is induced by increasing daylength is inhibited by biologically active sex steroids such as E2, T, and 11-KT.

Melatonin secretion from goldfish pineal gland in organ culture

Pineal glands were removed from goldfish reared under 12L-12D at 25 degrees for 2 weeks. These were incubated for 6 days under (1) normal 12L-12D (lights on 0600-1800 hr), (2) reversed 12L-12D (lights on 1800-0600 hr), (3) continuous dark, or (4) continuous light condition at 25 degrees. The incubation medium was changed at 12-hr intervals (0600-1800 and 1800-0600 hr) and secreted melatonin (MLT) was measured by RIA. Under 12L-12D or reversed 12L-12D, MLT secretion was active in the dark phase and was suppressed in the light phase of a given photoperiod. Under a continuous dark condition, a large amount of MLT was secreted into the medium, although the amount gradually decreased. The MLT secretion was more active in the period corresponding to the dark phase of the acclimatory photoperiod than in the period corresponding to the light phase. This pattern in secretion remained for 4 days. Under a continuous light condition, MLT secretion was suppressed, but the secretion was rapidly increased after changing the photoperiod from the light to the dark condition. These findings clearly indicate that MLT secretion in the organ-cultured pineal gland is photosensitive. It is active under dark and inactive under light conditions. The existence of a circadian rhythm in MLT secretion is also suggested.

Pheromone from ovulatory female goldfish induces gonadotropin surge in males

Characterization was made of the female stimuli which induce male gonadotropin (GtH) surge during spawning in the goldfish. In the presence of ovulatory females, olfactory tract sectioned (OTX) males failed to show sexual behavior or the GtH surge, while sham-operated (sham) males spawned and showed a GtH surge. When OTX and sham males were separated from ovulatory females with an opaque partition but with water circulating between male and female compartments, the GtH surge occurred only in sham males, although they could not court with the females. These results indicate that an olfactory stimulus (pheromone) from ovulatory females is essential to the occurrence of male GtH surge. Pheromone(s) from ovulatory female goldfish thus functions both as a releaser stimulating sexual behavior and as a primer inducing GtH surge in males.