GtH Surge Research Articles

Induction of Male-Type Gonadotropin Secretion by Implantation of 11-Ketotestosterone in Female Goldfish

In goldfish, plasma gonadotropin levels increase during spawning in both males and females (GTH surge). A female-typical GTH surge induces ovulation (ovulatory surge), and a male-typical surge triggers milt production in response to sex pheromones released from ovulatory females. This study examined whether the male-typical GTH surge occurs in adult females that are implanted with 11-ketotestosterone (KT), which induces male-typical sexual behavior in adult female goldfish. When KT-implanted females were exposed to ovulatory females, a GTH surge occurred without ovulation. No GTH surge was observed when KT-females were exposed to nonovulatory females. The GTH secretion in KT-females was further characterized by exposure to 17α,20β-dihydroxy-4-pregnen-3-one (17,20-P), a female sex pheromone that induces the GTH surge in males. Exposure to waterborne 17,20-P caused an elevation of GTH levels in KT-females as well as in males. The elevation of GTH levels induced by 17,20-P exposure was abolished when the KT-females were rendered anosmic. Unlike the female-typical ovulatory GTH surge that occurs in synchrony with photoperiod and peaks in the dark phase of the day, the 17,20-P-induced surge did not show a peak in the dark phase. These results indicate that the GTH surge in KT-females is a male-typical surge. Together with a previous study showing KT-induced behavioral masculinization (N. E. Stacey and M. Kobayashi, 1996,Horm. Behav.30,434–445), this adult gonochoristic species was shown to possess sexual plasticity of brain function in behavior and GTH secretion in response to sex steroid.

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.

Changes in brain levels of gonadotropin-releasing hormone and serum levels of gonadotropin and growth hormone in goldfish during spawning

Changes in serum levels of gonadotropin (GtH) and growth hormone (GH) and in levels of gonadotropin-releasing hormone (GnRH) in the pituitary and discrete brain areas were studied in male goldfish during spawning with spontaneously ovulating females. Spontaneous ovulation in females was induced by raising water temperature from 12 to 20 °C and providing spawning substrate of artificial floating vegetation. Spawning occurs naturally in sexually mature male goldfish in the presence of ovulating females. Serum GtH levels in spawning male goldfish exposed to ovulatory females increased markedly in synchrony with the ovulatory GtH surge in females. There was also a significant increase in serum GH levels in the spawning males. GnRH levels in the olfactory bulbs, telencephalon, hypothalamus, and pituitary of the spawning males showed marked decreases at the same time as serum levels of both GtH and GH peaked; these events in the males corresponded to the approximate time of ovulation, and similar changes in serum GtH and GH levels and brain GnRH levels, in the females. This temporal correlation between changes in GnRH levels in the brain and pituitary and increases in serum levels of GtH and GH in males as well as females supports the idea that activation of the GnRH neuronal system may be a common pathway for the stimulation of pituitary GtH and GH secretion in goldfish during spawning.

ＨＣＧ投与によるキンギョの排卵誘発時における血中ＧｔＨおよびステロイドホルモン濃度の変化

Changes in plasma gonadotropin (GtH) and steroid hormone levels were investigated during ovulation induced by human chorionic gonadotropin (HCG) in female goldfish. Sexually mature female goldfish maintained at 20°C under 14L10D (lights on at 0430hr) were injected with HCG at a dose of 10IU/g BW at 2100hr. Twenty-one fish out of 28 ovulated between 9 and 18 hours (0600 to 1500hr) after injection. In ovulatory fish, there was neither a significant increase nor surge in plasma GtH levels after the injection, whereas plasma testosterone and 17α, 20β-dihydroxy-4-pregnen-3-one levels showed changes similar to those occurring in spontaneous ovulation. These results suggest that injected HCG acts directly on the ovary without accelerating endogenous GtH secretion, and therefore induces ovulation in a similar manner as does the ovulatory GtH surge in spontaneous ovulation.

Steroid profiles during spawning in male common carp

The profiles of testosterone (T) 11-ketotestosterone (11-KT), 17α-hydroxy-4-pregnen-3-one (17-P), and 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) were determined in groups of spermiating male common carp ( Cyprinus carpio) which were or were not mated with females and subjected to an increase in water temperature, a treatment which usually induces females to spawn. The steroid levels were correlated with changes in gonadotropin (GtH). All males mated with ovulatory females showed a GtH surge and spawned. Two distinct classes of these spawned males were differentiated based on their initial androgen levels and hormone profiles. Androgen levels were initially elevated in the “high” males. In these fish there was a very rapid shift in the steroidogenic pathway from androgen to progestogen production coincident with the GtH surge. In the “low” males, 17-P levels rose significantly in response to the GtH surge, but there was no steroidogenic shift. In the males mated with nonovulatory females, the levels of 17-P, 17,20β-P, T, and 11-KT all increased coincident with a small rise in GtH; there was no steroidogenic shift. In the unmated males, the steroid profiles were similar to those in the nonspawned fish, except that T and 11-KT levels were significantly lower in comparison. In all groups, 17-P rose higher than 17,20β-P, and 11-KT was generally the predominant androgen. A model is proposed which explains the steroidogenic shift in the “high” males. This involves the inhibition of androgen biosynthesis by 17,20β-P or by a related progestogen.

Involvement of steroid hormones in the preovulatory gonadotropin surge in female goldfish

Goldfish,Carassius auratus, spawn several times within a spawning season. A gonadotropin (GtH) surge occurs at the time of ovulation in this cyprinid species. This ovulatory GtH surge mediates the processes of final oocyte maturation and ovulation, and occurs at the end of each spawning cycle. Within a cycle, there is a shift in the predominant plasma steroid from estradiol to testosterone, and finally 17α, 20β-dihydroxy-4-pregnen-3-one at the time of the ovulatory GtH surge. High levels of testosterone were always observed before ovulation. When ovariectomized or sexually regressed female goldfish were implanted with testosterone, they exhibited a GtH surge which was similar to those normally observed at ovulation. These results strongly suggest that elevated plasma testosterone is an important physiological requirement for the occurrence of the GtH surge.

Induction of gonadotropin surge by steroid hormone implantation in ovariectomized and sexually regressed female goldfish

Involvement of steroid hormones in the occurrence of the ovulatory gonadotropin (GtH) surge was studied in goldfish. Ovariectomized female goldfish were implanted with an empty Silastic capsule or a capsule containing testosterone or estradiol, and kept below 12° for 3 months (Experiment 1). Some of the steroid implanted fish showed a GtH surge which was quite similar to the normal ovulatory GtH surge in response to a water temperature rise from 12 to 20°, whereas no surge was observed in fish with empty capsules. When sexually regressed female goldfish were implanted with the capsules containing testosterone or estradiol and kept at 12° for 6 weeks out of spawning season, the GtH surge was also observed in these fish after the water temperature rise to 20° (Experiment 2). The GtH surge was observed in a larger number of testosterone-implanted fish than in the estradiol-treated fish in both experiments. These results strongly suggest that the high plasma level of testosterone observed before ovulation is an important physiological requisite for the occurrence of the ovulatory GtH surge in goldfish.

Effects of photoperiod and pinealectomy on the gonadotropin surge and ovulation in goldfish Carassius auratus.

Ovulation was induced in sexually mature female goldfish Carassius auratus maintained at 12°C under 15L-9D by raising the water temperature to 20°C under various photoperiods. The fish were checked every 2 or 3h, and those which had ovulated were blood-sampled to determine the timing of the GtH surge. In Experiment 1, 2 groups of fish (n=15 each) were placed under photoperiods fixed at 15L-9D. In Group A, lights on was 0400h, and in Group B, lights on was 1600h. Ovulation occurred during the dark-phase in both groups. In Experiment 2, the photoperiod was changed from 15L-9D (lights on 0400h) to continuous light (Group C; n=21) or continuous darkness (Group D; n=18) after the water temperature increase. In both groups, ovulations were observed at a time corresponding to the dark-phase of the acclimation photoperiod, however, in Group D, ovulation was slightly delayed when compared to Group C. In both Experiments, the GtH surge was observed only in ovulated fish. In Experiment 3, mature females were reared under 14L-10D (lights on 0430h) and either pinealectomized (PINX, Group E, n=19) or sham operated (Group F, n=21). The water temperature was increased one week following the operation. Fish from Group F ovualted during the dark-phase, whereas fish from Group E ovulated randomly. The GtH surge was observed in the ovulated fish from both groups.These results indicate that photoperiod plays an important role in determining the onset time of the preovulatory GtH surge. The pineal gland is somehow involved in the mechanism regulating this photoperiodic response.

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-17β (E 2) levels remained low for 3 days prior to ovulation. Postovulation E 2 levels that were significantly higher than the preovulation levels were kept elevated and declined on the third day after ovulation. These results indicate that E 2 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 E 2 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.

Correlation of the mating behaviour of bulls with patterns of gonadotrophins and oestrogens in oestrous cows

The correlation between mating behaviour of bulls and patterns of gonadotrophins (GTH, i.e. FSH and LH) and oestradiol-17β (E 2) was examined in oestrous cows, using two bulls. Four cows were placed with each bull in a free stall (10 m × 15 m) at 44 h after the second injection of PGF 2α analogue (cloprostenol). Mating (mounting and ejaculation) behaviour of the bull was observed and serum levels of FSH, LH and E 2 were measured in cows during the next 33 h. The bull ejaculated only with cows in which elevated E 2 levels and a GTH surge occurred. When the time of the GTH peak was designated as 0, and the patterns of hormones in cows with mating behaviour were combined, it was found that 92.3% of ejaculating and 79.1% of mounting behaviour occurred within the range of the GTH surge, indicating that the former behaviour was coincident with the GTH surge.

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°. 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 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-diOH-P) and testosterone showed a peak before ovulation, and then decreased by the time of ovulation. Estradiol-17β (E 2) showed moderate levels during the GtH surge. No marked decrease of E 2 levels was observed before the commencement of the GtH surge. Administration of testosterone or E 2 prior to elevating the water temperature did not inhibit the occurrence of the GtH surge and ovulation. Likewise, administration of neither 17α-hydroxyprogesterone nor 17α,20β-diOH-P affected plasma GtH levels at 20°. 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 E 2 levels triggers the GtH surge and ovulation.

Annual correlative changes in gonads and pituitary gonadotropes of feral African catfish, Clarias gariepinus

The reproductive cycle of male and female African catfish ( Clarias gariepinus) can be divided into a breeding period (May–August), a resting period (September–February) and a period of full gametogenesis (March–April). The pituitary gonadotropin (GTH) content and the ultrastructure of the gonadotropes largely parallel the cyclical changes in the gonads. The main characteristics of the pituitary GTH cycle are a prespawning GTH surge and a postspawning regression of the gonadotropes. These phenomena are suppressed when African catfish are kept under favourable husbandry conditions. Under such circumstances the pituitary stores large amounts of GTH and shows a limited, continuous secretion of the hormone, sufficient for a sustained gametogenesis and gonadal steroid production, but not for spontaneous spawning. Under such adequate fish culture conditions, ovulation can be induced and healthy larvae obtained at any time of year.

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 occurence 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.

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 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-diOH-P) in males, and for GtH in females. Spawning was induced by raising water temperature from 12 to 20° 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 17α,20β-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.

コイの排卵過程における血中GtHおよびステロイドホルモン量の変化

Plasma gonadotropin (GtH) and steroid hormone levels were monitored during ovulation in the common carp Cyprinus carpio. Nine out of 14 females ovulated after the water temperature was increased from 16 to 24°C. In the ovulated females, plasma GtH showed acute synchronous elevations in the afternoon around 1500 hours, peaked at midnight, and returned to the basal levels in the morning. Ovulationw as observed at midnight and early morning. Plasma 17α-hydroxy-progesterone and 17α, 20β-dihydroxy-4-pregnen-3-one fluctuated in phase with GtH. Only small changes were observed in the plasma E2 levels during ovulation. In most of the ovulated fish, testosterone distinctly increased with the commencement of temperature elevation and remained at high levels during the GtH surge, whereas in other ovulated fish only acute changes following the GtH surge were observed. In the unovulated females, large peaks of plasma GtH and steroid hormones were not observed. In some females, however, small rhythmical or irregular changes in GtH and/or steroid hormone levels were observed.

Photoperiodic determination of preovulatory gonadotropin surge onset time in the carp Cyprinus carpio.

Four experiments were conducted to clarify the effects of photoperiod on the onset time of the preovulatory GtH surge in the female carp Cyprinus carpio. After acclimation together with males under photoperiods of 12L-12D (Group A, lights-on 0600-1800 hours; Group B, 1800-0600 hours) in Experiment 1, 18L-6D (Group C, lights-on 0000-1800 hours; Group D, 0600-2400 hours) in Experiment 2, and 16L-8D (lights-on; 1600-0800 hours) in Experiments 3 (Group E and F) and 4 (Group G and H), water temperature was raised from 16 to 24°C beginning at 1730 hours to induce ovulation and spawning. In Experiments 1 and 2, a preovulatory GtH surge initiated in the latter part of the light-phase in each photoperiod group after the water temperature was increased irrespective of the starting time of temperature elevation, and ovulation was observed mainly during the dark-phase. In Experiment 3, the light-phase period was changed in Group F from 1600-0800 to 0400-2000 hours after increasing the temperature so that the dark-phase was abruptly inserted after a 4-h light-phase. This change resulted in a delay of the preovulatory GtH surge. In Experiment 4, the GtH surge began at the same clock time as in Experiments 1 and 2 corresponding to the latter part of the light-phase of the acclimation photoperiod even though the photoperiod was changed to 24L and 24D in Group G and H, respectively, after increasing water temperature. These results indicate that female carp have a reproductive circadian rhythm and the onset time of the preovulatory GtH surge is determined by a photoperiodic cue.

Radioimmunoassay for silver carp gonadotropin.

A radioimmunoassay for cyprinid gonadotropin (GtH) was developed using GtH purified from the silver carp Hypophthalmichthys molitrix and its specific antiserum. The minimum detectable quantity of silver carp GtH was 0.58ng/ml (29pg/tube). The intraassay and the inter-assay coefficients of variation were 6.8% (N=6) and 8.6% (N=5), respectively near 50% binding. This system was found to be suitable for the measurement of plasma GtH of goldfish Carassius auratus and carp Cyprinus carpio and pituitary GtH of some teleosts. The competition curves for goldfish and carp plasma were parallel to the standard curve with silver carp GtH. Pituitary extracts of goldfish, carp, honmoroko Gnathopogon elongatus, medaka Oryzias latipes, and chichibu goby Tridentiger obscurus showed competition curves parallel to the silver carp GtH standard, but plasma and pituitary samples of bitterlings (Cyprinidae) showed less steep curves than the standard. This system was physiologically validated by detecting the ovulatory GtH surge in individual goldfish. Plasma steroid hormone levels during ovulation were also measured and their role were discussed in relation to the GtH surge.

The induced spawning of roach, Rutilus rutilus (L.), with the antioestrogens clomiphene and tamoxifen

The induced spawning of gravid roach was investigated using the antioestrogens clomiphene and tamoxifen. Three dose levels ofeach were used: 0·1, l or 10 mgkg‐1, given twice with a 4‐day interval to groups of eight fish with saline controls. Running males were randomly distributed. Tamoxifen, when injected at a rate of l mg kg‐1 was found to be most successful. This treatment induced ovulation in five of the six females, and profuse spawning on 3 consecutive days, 4 days after the first injection. Clomiphene induced ovulation and spawning in one of six females at 2 × 10 mg kg‐1, and two of eight females at 2 × l mg kg‐1, respectively 6 and 7 days after the first injection. The eggs produced showed normal devel‐opment. No control fish ovulated or spawned. Both drugs probably act by indirect mechanisms, blocking sex steroid feedback inhibition of gonadotropin (GtH) secretion at the pituitary, thereby inducing a plasma GtH surge. The results of this experiment suggest that tamoxifen may be an effective substitute for pituitary preparations in the induced spawning of fish.

The ovulatory surge of gonadotropin and sexual receptivity in the female golden hamster

The present experiments were undertaken to study the nature of the relationship between ovulatory gonadotropin (GTH) release and subsequent sexual behavior in the hamster. GTH antiserum (GTH A/S) injected just prior to the release of the GTH surge prevented both ovulation and lordotic behavior. Both behavior and ovulation could be restored by injecting LH. FSH was effective in restoring ovulation but appeared to have less effect on behavior as compared to LH. Progesterone restored behavior but not ovulation. Large doses of estradiol had no effect on either parameter. The results suggest that ovulatory GTH release (especially LH) is a central event leading to the onset of a sexual receptivity. GTH probably acts by stimulating ovarian progesterone. This steroid acts to induce receptivity and also appears to induce a subsequent period during which the female is behaviorally refractory to sex steroids.