GTH Secretion Research Articles

A novel agent for induced spawning using a combination of GnRH analog and an FDA-approved dopamine receptor antagonist

In certain fish species, endogenous dopamine has a significant inhibitory effect on LH release and spawning induced by externally applied GnRH. Preovulatory LH surge and spawning in these fish species are caused by the simultaneous release of GnRH and exclusion of the dopaminergic inhibition. To induce spawning, a potent dopamine D2 receptor (DRD2) antagonist and a GnRH analog are concomitantly applied to fish with solid dopaminergic inhibition. However, the currently used dopamine antagonists are not approved by the FDA for veterinary use. This project was conducted in order to find a novel FDA-approved dopamine antagonist that will be efficient in spawning induction in fish species and will not be lethal to the fish. First, we studied the inhibitory effect of the novel dopamine antagonist, azaperone, on tilapia DRD2, transiently expressed in COS-7 cell line. Azaperone was more effective than metoclopramide in antagonizing the effect of quinpirole on taDRD2 both in terms of EC50 and maximal response. Next, we tested the ability of the combination of the dopamine antagonist and sGnRHa on GTH secretion, in adult tilapia. The novel dopamine antagonist significantly increased the GnRH-stimulated LH release in vivo, on both LH release and gene expression. We next aimed to test the novel combination in carp. The combination of the novel dopamine antagonist together with GnRHa, increased the release of estradiol and LH, in a dose-dependent manner. A higher hatching rate and spawning efficiency were observed than in the positive control that contained metoclopramide as a dopamine antagonist.

Identifying the Interaction of the Brain and the Pituitary in Social – and Reproductive – State of Tilapia by Transcriptome Analyses

Introduction: As in all vertebrates, reproduction in fish is regulated by gonadotrophin-releasing hormone (GnRH) control on gonadotrophic hormones (GtHs) activity. However, the neuroendocrine factors that promote GnRH and GtH activity are unknown. In Nile tilapia (Oreochromis niloticus), sexual activity and reproduction ability depend on social rank; only dominant males and females reproduce. Here, this characteristic of dominant fish allows us to compare brain and pituitary gene expression in animals that do and do not reproduce, aiming to reveal mechanisms that regulate reproduction. Methods: An extensive transcriptome analysis was performed, combining two sets of transcriptomes: a novel whole-brain and pituitary transcriptome of established dominant and subordinate males, together with a cell-specific transcriptome of luteinizing hormone (LH) and follicle-stimulating hormone cells. Pituitary incubation assay validated the direct effect of steroid application on chosen genes and GtH secretion. Results: In most dominant fish, as determined behaviorally, the gonadosomatic index was higher than in subordinate fish, and the leading upregulated pituitary genes were those coding for GtHs. In the brain, various neuropeptide genes, including isotocin, cholecystokinin, and MCH, were upregulated; these may be related to reproductive status through effects on behavior and feeding. In a STRING network analysis combining the two transcriptome sets, brain aromatase, highly expressed in LH cells, is the most central gene with the highest number of connections. In the pituitary incubation assay, testosterone and estradiol increased the secretion of LH and specific gene transcription. Conclusions: The close correlation between behavioral dominance and reproductive capacity in tilapia allows unraveling novel genes that may regulate the hypothalamic-pituitary-gonadal axis, highlighting aromatase as the main factor affecting the brain and pituitary in maintaining a sexually active organism.

Effects of LPXRFamide peptides on chub mackerel gonadotropin secretion†.

Gonadotropin-inhibitory hormone (GnIH), a neuropeptide, suppresses gonadotropin (GTH) secretion in birds and mammals. In fish, the GnIH homolog LPXRFamide (LPXRFa) produces mature peptides with species-dependent effects on sexual reproduction. Here, we investigated the effects of LPXRFa on GTH secretion in the chub mackerel (cm; Scomber japonicus). We cloned cmlpxrfa (603bp) and cmlpxrfa-r (1416bp). Additionally, we isolated lpxrfa from the bluefin tuna (Thunnus orientalis) to confirm the conservation of the LPXRFa mature sequence. Phylogenetic analysis showed that the LPXRFa precursor protein produces three mature peptides, LPXRFa-1, -2, and -3, in both species. Reverse transcription-quantitative polymerase chain reaction revealed that cmlpxrfa is expressed in the hypothalamus and thalamus and midbrain (T.MB), and sexual differences were observed. Receptor expression was observed in the preoptic area, hypothalamus, T.MB, and pituitary. Female hypothalamic lpxrfa expression did not change during puberty. Reporter gene assay showed that LPXRFa induced receptor activation via the CRE and SRE signaling pathways. However, in the presence of forskolin, an intracellular cyclic AMP enhancer, none of the LPXRFa could suppress receptor activity. The in vitro bioassay results showed that gonadotropin-releasing hormone-1 (GnRH1) had no effect on follicle-stimulating hormone (FSH) secretion, whereas the three LPXRFa significantly increased FSH secretion in pituitary cells from male chub mackerel. Contrarily, GnRH1 and three LPXRFa significantly increased luteinizing hormone secretion. The in vivo administration of LPXRFa had no effect on fshb and lhb expression in prepubertal and mature male chub mackerel. Overall, cmLPXRFa lacks the ability to suppress GTH secretion but can promote GTH secretion.

In vitro action of leptin on gonadotropin secretion in pre-pubertal male chub mackerel

Leptin directly influences gonadotropin (GTH) secretion from female pituitary cells in vitro and is a key signal at the onset of puberty in female chub mackerel (Scomber japonicus). Here, we investigated whether leptin also influences GTH secretion in male chub mackerel. The addition of 1 nM homologous recombinant leptin to pre-pubertal male pituitary cells stimulated follicle-stimulating hormone secretion after 1 and 2 h of culture. Therefore, leptin signaling could also directly facilitate GTH secretion in male chub mackerel.

LPXRFa, the piscine ortholog of GnIH, and LPXRF receptor positively regulate gonadotropin secretion in Tilapia (Oreochromis niloticus).

LPXRFamide (LPXRFa) peptides have been characterized for their ability to inhibit gonadotropin (GTH) release in birds and stimulate growth hormone (GH) release in frogs. However, their involvement in regulating the reproductive hypothalamo-pituitary-gonadal axis in mammals and fish is inconclusive. To study the role of LPXRFa peptides in the regulation of GTH secretion, we cloned tilapia LPXRFa and LPXRF receptor (LPXRF-R). Processing of the tilapia preproLPXRFa liberated three mature LPXRFa peptides that varied in size and post-translational modifications. Phylogenetic analysis of LPXRFa and the closely related RFamide peptide PQRFa showed clear clustering of each peptide sequence with its orthologs from various vertebrates. Signal-transduction analysis of the tilapia LPXRF-R in COS-7 cells showed clear stimulation of CRE-dependent luciferase activity, whereas the human NPFFR1 showed suppression of forskolin-induced CRE-dependent activity in this system. Administration of the tilapia pyroglutaminated LPXRFa-2 peptide to primary cell culture of tilapia pituitaries, or to reproductive female tilapia by ip injection, positively regulated both LH and FSH release in vivo and in vitro. Using double-labeled fluorescent in-situ hybridization and immunofluorescence, βLH cells were found to co-express both tilapia lpxrf and tilapia lpxrf-r mRNA, whereas some of the βFSH cells coexpressed only lpxrf-r mRNA. No coexpression of tilapia lpxrf-r was identified in GH-positive cells. These findings suggest that the LPXRFa system is a potent positive regulator of the reproductive neuroendocrine axis of tilapia.

Effects of estradiol or testosterone treatment on expression of gonadotropin subunit mRNAs and proteins in the pituitary of juvenile brown hagfish, Paramyxine atami

A single functional gonadotropin (GTH) comprising two subunits, α and β, was recently identified in the pituitary of brown hagfish (Paramyxine atami). Little is known about the feedback mechanisms that regulate these GTH subunits by sex steroids in the hagfish. The present study was designed to examine feedback effects of estradiol and testosterone on mRNA expression and protein expression of both GTHα and GTHβ subunits in the pituitary of the juvenile P. atami. Intraperitoneal administration of estradiol over the course of 27days resulted in substantial accumulation of immunoreactive (ir)-GTHα and ir-GTHβ in the adenohypophysis, but testosterone treatments over 27days had no effects on ir-GTHα or ir-GTHβ. Estradiol treatment for 1, 2, 4 or 14days had no effect on GTHα mRNA levels. In contrast, after 2days of estradiol treatment, GTHβ mRNA levels had increased significantly from baseline, while these levels were not affected after treatment over 1, 4, or 14days. After 14days of testosterone treatment, both GTHα and GTHβ mRNA levels had decreased significantly from baseline levels. These results indicate that estradiol acted primarily to suppress the secretion of GTH, and hence resulted in the accumulations of ir-GTHα and ir-GTHβ in the pituitary. On the other hand, testosterone appeared to suppress both the synthesis and the secretion of GTH. Thus, estradiol and testosterone probably differ in their effects on the regulation of pituitary GTH synthesis and secretion in juvenile hagfish.

Sex Steroid Levels and Breeding Performance of F1 Generation Malaysian Mahseer, Tor tambroides (Bleeker, 1854) by Removal of Dopaminergic Inhibition

Induced ovulation of captive Malaysian mahseer (Tor tambroides) often encounters low reproductive performances. The present study was conducted to gain insight to problems associated with poor breeding performance of Malaysian mahseer in captivity. In this study, sex steroid hormone levels and ovulation performance in filial 1 (F1) generation of Malaysian mahseer (Tor tambroides) were investigated. Fishes were injected with ovatide, combination of Salmon Gonadotropin Releasing Hormone Analogue (sGnRHa), DOM, sGnRHa+DOM and 0.9% NaCl. A total of 30 matured females T. tambroides with weight ranged from 2.01-3.80 kg were used in this study. The females were given ovaplant (39.5-37.3 μg kg-1) pretreatment for 6 weeks prior to hormones injection. Females were given a single intramuscular injection of ovatide (0.5 mL kg-1 b.wt.), sGnRHa (10 μg kg-1 b.wt.), DOM (5 mg kg-1 b.wt.), sGnRHa+DOM (10 μg kg-1 b.wt.+5 mg kg-1 b.wt.) or 0.9% NaCl. Blood samples were collected at 0, 6, 12 and 24 h (s) after injection. The effect of these treatments on sex steroids level (Testosterone (T) and 17β-Estradiol (E2) and ovulation performance of F1 Tor tambroides were evaluated. Result showed that higher ovulatory response was observed in the group treated with positive control ovatide (0.5 mL kg-1 b.wt.). Interestingly, it was observed that sGnRHa and Domperidone (DOM) produce highest egg and larval qualities compared to ovatide and sGnRHa alone. No ovulation occurred in the group treated with DOM alone. Sex steroids plasma concentration of T and E2 increased significantly in association with ovulation. The levels fluctuated and reached a peak at 12 h, then dropped dramatically at 24 Post Injection (PI). In contrast the non-ovulated groups, the sex steroids hormone concentration showed little increment after injection and rose at 12 h PI, then slowly dropped at 24 h pi. These results provide evidence for a strong dopamine inhibition on GtH secretion in captive mahseer.

Evolutionary Origin of the Structure and Function of Gonadotropin-Inhibitory Hormone: Insights from Lampreys

Gonadotropin (GTH)-inhibitory hormone (GnIH) is a novel hypothalamic neuropeptide that inhibits GTH secretion in mammals and birds by acting on gonadotropes and GnRH neurons within the hypothalamic-pituitary-gonadal axis. GnIH and its orthologs that have an LPXRFamide (X = L or Q) motif at the C terminus (LPXRFamide peptides) have been identified in representative species of gnathostomes. However, the identity of an LPXRFamide peptide had yet to be identified in agnathans, the most ancient lineage of vertebrates, leaving open the question of the evolutionary origin of GnIH and its ancestral function(s). In this study, we identified an LPXRFamide peptide gene encoding three peptides (LPXRFa-1a, LPXRFa-1b, and LPXRFa-2) from the brain of sea lamprey by synteny analysis and cDNA cloning, and the mature peptides by immunoaffinity purification and mass spectrometry. The expression of lamprey LPXRFamide peptide precursor mRNA was localized in the brain and gonad by RT-PCR and in the hypothalamus by in situ hybridization. Immunohistochemistry showed appositions of lamprey LPXRFamide peptide immunoreactive fibers in close proximity to GnRH-III neurons, suggesting that lamprey LPXRFamide peptides act on GnRH-III neurons. In addition, lamprey LPXRFa-2 stimulated the expression of lamprey GnRH-III protein in the hypothalamus and GTHβ mRNA expression in the pituitary. Synteny and phylogenetic analyses suggest that the LPXRFamide peptide gene diverged from a common ancestral gene likely through gene duplication in the basal vertebrates. These results suggest that one ancestral function of LPXRFamide peptides may be stimulatory compared with the inhibitory function seen in later-evolved vertebrates (birds and mammals).

Intrahepatic lithiasis

Leptin directly influences gonadotropin (GTH) secretion from female pituitary cells in vitro and is a key signal at the onset of puberty in female chub mackerel (Scomber japonicus). Here, we investigated whether leptin also influences GTH secretion in male chub mackerel. The addition of 1 nM homologous recombinant leptin to pre-pubertal male pituitary cells stimulated follicle-stimulating hormone secretion after 1 and 2 h of culture. Therefore, leptin signaling could also directly facilitate GTH secretion in male chub mackerel.

Role of LPXRFamide peptide in the neuroendocrine regulation of reproduction in fish

Abstract The decapeptide gonadotropin-releasing hormone (GnRH) is the primary factor responsible for the hypothalamic control of gonadotropin (GTH) secretion. This review focuses on a family of neuropeptides, LPXRFamide (LPXRFa) peptides, which have been implicated in the regulation of GTH secretion. LPXRFa acts on the pituitary via a G protein-coupled receptor, LPXRFa-R, to enhance gonadal development and maintenance by increasing gonadotropin release and synthesis. Because LPXRFa exists and functions in several fish species, LPXRFa is considered to be a key neurohormone in fish reproduction control. The precursors to LPXRFamide peptides encoded plural LPXRFamide peptides and were highly divergent in vertebrates, particularly in lower vertebrates. Tissue distribution analyses indicated that LPXRFamide peptides were highly concentrated in the hypothalamus and other brainstem regions. In view of the localization and expression of LPXRFamide peptides in the hypothalamo-hypophysial system, LPXRFamide peptide in fish increase GTH release in vitro and in vivo. This review summarizes the advances made in our understanding of the biosynthesis, mode of action and functional significance of LPXRFa, a newly discovered key neurohormone.

A review of the control of reproduction and hormonal manipulations in finfish species

Control of reproductive is essential for the sustainability of commercial aquaculture production. The reasons for breakage of ovulation and spawning is failure of the pituitary to release gonadotropin hormones. Gonadotropin hormones secretion is under a dual control, by gonadotropin releasing hormone (GnRH), and inhibition by dopamine on the actions of GnRH. The inhibitory actions of dopamine on GTH secretion can vary in potency between species. In some fish it is necessary to use dopamine blocker, such as domperidone, results in potentiation of the actions of GnRH analoge, leading to a large release of GTH-II and ovulation. In some other injection of a high dose of GnRH analoge alone is effective. In several marine species GTH-II secretion is not under dopaminergic inhibitory control but sensitive to exogenous analoge hormones (GnRHa). In many fishes, synthetic analoges of lutenizing hormone-releasing hormone (LHRHa) are widely used for inducing spawning. In several tropical freshwater and marine finfishes the human chorionic gonadotropin (HCG), pituitary homogenate and semi-purified fish gonadotropin are used. Rrecently the replacement of natural GnRH are used and had resulted in a centuple increase in the effectiveness of luteinizing hormones secretion induction. Key word: Reproduction induced spawning, dopamine antagonists, GTH-II- HCG

Oral contraceptives decrease saliva testosterone but do not affect the rise in testosterone associated with athletic competition

Women athletes from intercollegiate soccer, volleyball, and softball teams, and women skaters from a team competing in an amateur roller derby league, contributed saliva samples before warm-up and immediately after the completion of one or more sanctioned competitions. Women using oral contraceptives (OCs, n = 29) had a significantly lower mean level of saliva testosterone (T) than non-users ( n = 51). Thus, OCs contribute predictable variation to individual differences in saliva T, and OC use is likely to contribute to individual differences in measures of psychological processes and/or behavior which are causally related to individual differences in circulating testosterone. Most of the women ( n = 68) played during one or more of the competitions for which they contributed saliva samples. Whether for soccer, volleyball, softball, or roller derby, competition was associated with a robust increase in saliva T. Although OC users had significantly lower saliva T levels than non-users before and after-competition, both users and non-users showed virtually the same increase in saliva T over the course of competition. While the most proximal cause of this increase is not known, it is probably not the result of an increase in gonadotropin (GTH) secretion since an increase in GTH secretion would presumably be prevented by OC use.

Time course of final oocyte maturation and ovulation in chub mackerelScomber japonicusinduced by hCG and GnRHa

The question of whether the ovulation and spawning time in chub mackerel Scomber japonicus is entrained by a circadian rhythm was raised by our previous experiments. Further questions were also raised about whether the time course of human chorionic gonadotropin (hCG)-induced final oocyte maturation (FOM) and ovulation reflected the natural time course induced by endogeneous pituitary gonadotropin (GtH). To address these questions, hCG and gonadotropin-releasing hormone analog (GnRHa) were administered at two ‘opposite’ times, 14:00 and 02:00 hours, and the time courses of FOM and ovulation were compared. When hCG was injected, ovulation occurred 33 h post-injection in both groups, regardless of the timing of the hCG injection. The timing of ovulation in chub mackerel depends on the timing of hCG injection, but apparently not on circadian rhythms. When GnRHa was injected, ovulation began at 36 h post-injection of GnRHa, regardless of the timing of injection. These results indicate that the time course of FOM and ovulation in the chub mackerel followed a similar pattern whether stimulated by hCG injection or spontaneous luteinizing hormone (LH) surge because GnRHa induces the secretion of endogenous GtH (primarily LH) from the fish pituitary. Thus, it is concluded that the time course of hCG-induced FOM and ovulation in chub mackerel follows the natural time course induced by endogenous pituitary LH.

Sexually dimorphic expression of pituitary glycoprotein hormones in a sex‐changing fish (Pseudolabrus sieboldi)

It is widely accepted that the hypothalamic-pituitary-gonadal axis is involved in gonadal sex change in socially controlled sex-changing fish. However, the specific secretion profiles of pituitary gonadotropins (GtHs) in this type of fish are not known. To address this fundamental question, we demonstrated that the diurnal secretion patterns of GtHs differ distinctly between males and females in a socially controlled sex-changing fish. We analyzed the pituitary mRNA levels of glycoprotein hormone subunits (i.e., the common alpha-subunit and specific beta-subunits follicle-stimulating hormone beta, luteinizing hormone beta, and thyroid-stimulating hormone beta) in the wrasse Pseudolabrus sieboldi, which is a model fish that exhibits accurate diurnal rhythms of gametogenesis in both males and females. Northern blots clearly showed that each subunit gene exhibits a diurnal rhythm of expression in the pituitary and that the expression patterns differ distinctly between the sexes. Our results suggest that oogenesis and spermatogenesis in this hermaphroditic fish are regulated differentially through the distinct secretion patterns of pituitary glycoprotein hormones. This study also provides direct evidence of the sexual plasticity of pituitary GtH secretion in a socially controlled sex-changing fish.

Effects of a gonadotropin‐releasing hormone antagonist on gonadotropin levels in masu salmon and sockeye salmon

The salmon gonadotropin-releasing hormone (sGnRH) is considered to be involved in gonadal maturation via gonadotropin (GTH) secretion in salmonid fishes. However, there is no direct evidence for endogenous sGnRH-stimulated GTH secretion in salmonids. In this study, to clarify whether endogenous sGnRH stimulates GTH secretion, we examined the effects of the mammalian GnRH (mGnRH) antagonist [Ac-Delta(3)-Pro(1), 4FD-Phe(2), D-Trp(3,6)]-mGnRH on luteinizing hormone (LH) levels in 0-year-old masu salmon Oncorhynchus masou and sockeye salmon Oncorhynchus nerka. First, the effects of the GnRH antagonist on LH release were examined in 0-year-old precocious male masu salmon. GnRH antagonist treatment for 3 hr significantly inhibited an increase in plasma LH levels that was artificially induced by exogenous sGnRH administration, indicating that the GnRH antagonist is effective in inhibiting LH release from the pituitary. Subsequently, we examined the effect of the GnRH antagonist on LH synthesis in 0-year-old immature sockeye salmon that were pretreated with exogenous testosterone for 42 days to increase the pituitary LH contents; the testosterone treatment did not affect the plasma LH levels. GnRH antagonist treatment slightly but significantly inhibited an increase in the testosterone-stimulated pituitary LH content levels. However, no significant differences in the plasma LH levels were observed between the GnRH antagonist-treated and control groups. These results suggest that endogenous sGnRH is involved in LH secretion in salmonid fishes.

Role of PKC in the regulation of gonadotropin subunit mRNA levels: interaction with two native forms of gonadotropin-releasing hormone

Gonadotropin-releasing hormone (GnRH) is an important regulator of reproduction in all vertebrates through its actions on the production and secretion of pituitary gonadotropin hormones (GtHs). Most vertebrate species express at least two GnRHs, including one form, designated chicken (c)GnRH-II or type II GnRH, which has been well conserved throughout evolution. The goldfish brain and pituitary contain salmon GnRH and cGnRH-II. In goldfish, GnRH-induced luteinizing hormone (LH) secretion involves PKC; however, whether PKC mediates GnRH stimulation of GtH subunit mRNA levels is unknown. In this study, we used inhibitors and activators of PKC to examine its possible involvement in GnRH-induced increases in GtH-alpha, follicle-stimulating hormone (FSH)-beta and LH-beta mRNA levels in primary cultures of dispersed goldfish pituitary cells. Treatment with PKC inhibitors calphostin C and GF109203X unmasked a basal repression of GtH subunit mRNA levels by PKC; both inhibitors increased GtH subunit mRNA levels in a dose-dependent manner. PKC activators, 12-O-tetradecanoylphorbol 13-acetate (TPA), and 1,2-dioctanoyl-sn-glycerol, stimulated GtH subunit mRNA levels, whereas an inactive phorbol ester (4-alpha-TPA) was without effect. Thus, a dual, inhibitory and stimulatory, influence for PKC in the regulation of GtH subunit mRNA levels is suggested. In contrast, PKC inhibitor- and activator-induced effects were, for the most part, additive to those of GnRH, suggesting that conventional and novel PKCs are unlikely to be involved in GnRH-stimulated increases in GtH subunit mRNA levels. Our data illustrate major differences in the signal transduction of GnRH effects on GtH secretion and gene expression in the goldfish pituitary.

Seasonal dynamics in gonadotropin secretion and E2-binding in the catfish Heteropneustes fossilis

In the present investigation, significant annual/seasonal variations were noticed in plasma and pituitary gonadotropin (GTH) which were correlated with gonado-somatic index, plasma estradiol-17beta, and nuclear E(2) receptor (NE(2)R) in the pituitary, hypothalamus and telencephalon. The NE(2)R concentrations and dissociation constant (k (d)) values showed significant seasonal variations with high values in the late preparatory phase and low values in the postspawning phase. The NE(2)R levels were the highest in the pituitary, followed by the hypothalamus and telencephalon in all the seasons. In the prespawning phase, ovariectomy (OVX) elicited a strong negative feedback on GTH secretion with a bimodal pattern of release and elevated the NE(2)R levels and k (d) values, without producing any significant change in the resting phase suggesting that E(2) appears to exert differential feedbacks on GTH secretion.

Lunar cycles and reproductive activity in reef fishes with particular attention to rabbitfishes

AbstractCues from the moon influence synchrony in growth, feeding, migration, behaviour and reproduction of many reef fishes. Compared with comprehensive studies on the annual and daily activities of fish, few physiological studies have paid attention to the importance of lunar cues in reproductive activities. We review mutual and interesting relationships between fish reproduction and environmental changes induced by the moon, with particular emphasis on the reproductive activity of the rabbitfishes (Siganidae). Rabbitfish species exhibit, in nature, a definitive reproductive season, which differs among the tropical areas. During the reproductive season, synchronous spawning of rabbitfish is associated with a particular lunar phase. The lunar phase used by the respective species is similar in different regions on the earth. Histological observations revealed that gonads develop synchronously towards a peak around the spawning lunar phase, after which the gonads return to spent condition. Concomitant with gonadal development, sex steroid hormones were produced under the influence of gonadotropin (GtH). Injections of human chronic gonadotropin (hCG) to the fish that are undergoing active spermatogenesis accelerated testicular maturation. These results suggest that hormonal response in maturing the gonads in rabbitfish is under the regulation of GtH, and that pituitary secretion of GtH according to the lunar cycle accounts for the lunar rhythm in gonadal development. We speculate that the cues from the moon can be recognized by the higher parts of the hypothalamus–pituitary–gonadal axis. Possible relationships between exogenous environmental factors and the lunar‐reproductive rhythm are also discussed.

Evidence for a novel intrapituitary autocrine/paracrine feedback loop regulating growth hormone synthesis and secretion in grass carp pituitary cells by functional interactions between gonadotrophs and somatotrophs.

Gonadotropin (GTH) and GH released from the pituitary are known to interact at multiple levels to modulate the functions of the gonadotrophic and somatotrophic axes. However, their interactions at the pituitary level have not been fully characterized. In this study, autocrine/paracrine regulation of GH synthesis and secretion by local interactions between gonadotrophs and somatotrophs was examined using grass carp pituitary cells as a cell model. Exogenous GTH and GH induced GH release and GH mRNA expression in carp pituitary cells. Removal of endogenous GTH and GH by immunoneutralization with GTH and GH antisera, respectively, suppressed GH release, GH production, and GH mRNA levels. GH antiserum also blocked the stimulatory effects of exogenous GTH on GH release and GH mRNA levels. In reciprocal experiments, GH release and GH mRNA expression induced by exogenous GH was significantly reduced by GTH antiserum. In addition, exogenous GH was found to be inhibitory to basal GTH release and treatment with GH antiserum elevated GTH secretion at low doses but suppressed GTH production at high doses. These results suggest that local interactions between gonadotrophs and somatotrophs may form an intrapituitary feedback loop to regulate GH release and synthesis. In this model, GTH released from gonadotrophs induces GH release and GH production in neighboring somatotrophs. GH secreted maintains somatotroph sensitivity to GTH stimulation, and at the same time, inhibits basal GTH release in gonadotrophs. This feedback loop may represent a novel mechanism regulating GH release and synthesis in lower vertebrates.

Induction of ovulation in endemic pearl mullet ( Chalcalburnus tarichi), living in the highly alkaline Lake Van, using GnRHa ([ d-Ser( tBu) 6,Pro 9-Net]-GnRH) combined with haloperidol

Pearl mullet ( Chalcalburnus tarichi) (Cyprinidae) is an endemic fish species of the Lake Van (Turkey), the largest soda lake (pH 9.8) on earth. During April–June, the spawning period of the fish, the mullet migrate from Lake Van to freshwater rivers around the lake and after spawning they return to the lake. This fish gives hope for improving the fishery in the alkaline environment deprived of other commercial fish species. The objective of this study was to find an optimal method for inducing ovulation of pearl mullet. Mammalian GnRH analogue (GnRHa at 60 μg/kg); dopamine antagonist, haloperidol (HAL at 1.5 mg/kg) and a combination of GnRHa and haloperidol (GnRHa+HAL) at low (20 μg/kg GnRHa and 0.5 mg/kg HAL) and high (60 μg/kg GnRHa and 1.5 mg/kg HAL) doses were tested for this purpose. Saline (0.7% NaCl) injected fish were used as a control group and no ovulation occurred in this group. Based on spawning ratio (the proportion of fish that spawned in each group), spawning index (the weight of eggs as a percentage of female body weight) and fertilization rate, the GnRHa+HAL combination at the high (60 μg/kg GnRHa and 1.5 mg/kg HAL) doses were found to be more efficient (7/7, 16.0±2.4, 86.0±10 respectively) than the low (20 μg/kg GnRHa and 0.5 mg/kg HAL) doses of GnRHa+HAL (3/7, 12.4±2.1, 54.5±6.5 respectively). No females ovulated in the treatment group receiving HAL at 1.5 mg/kg and GnRHa alone at 60 μg/kg. It is concluded that GnRHa+HAL (60 μg/kg+1.5 mg/kg) treatment is effective and reliable for induction of ovulation in pearl mullet broodstocks. Haloperidol improved the GnRHa effect on ovulation. GnRHa alone is not sufficient for spawning induction in the pearl mullet. The results indicate that in pearl mullet, similar to other cyprinids, there is a strong dopamine inhibitory tone on GtH secretion. In addition, the ovulation index and fertilization rates of eggs were influenced by the dosages of the applied GnRHa+HAL combination.