Differential distribution and response to experimental sexual maturation of two forms of brain gonadotropin-releasing hormone (GnRH) in the European eel,Anguilla anguilla

Using specific radioimmunoassays for the two GnRH molecular forms present in the European eel, Anguilla anguilla, (mGnRH and cGnRH II), we compared their distributions in the pituitary and different parts of the brain of female silver eels, as well as the modifications of their levels in experimentally matured female eels (treated with carp pituitary extract). In control eels, mGnRH levels were higher than cGnRH II levels in the pituitary, olfactory lobes and telencephalon, di- and mesencephalon, while the opposite was found in the posterior part of the brain (met- and myelencephalon). Experimental sexual maturation of the gonads significantly increased mGnRH levels in the pituitary and anterior parts of the brain; such a positive effect was not observed on the low cGnRH II levels, which were, in contrast, reduced. These data indicate that the positive feedback of gonadal hormones on GnRH, that we previously demonstrated, would specifically affect the mGnRH form. The differential distribution and control of mGnRH and cGnRH II suggest that these two forms have different physiological roles in the eel. The large increase in mGnRH during sexual maturation suggests the prime implication of this form in the neuroendocrine control of reproduction.     

Pituitary gonadotropin-releasing hormone (GnRH) receptor activity in goldfish and catfish: seasonal and gonadal effects

The goldfish pituitary contains two classes of gonadotropin-releasing hormone (GnRH) binding sites, a high affinity/low capacity site and a low affinity/high capacity site (Habibiet al. 1987a), whereas the catfish pituitary contains a single class of high affinity GnRH binding sites (De Leeuwet al. 1988a). Seasonal variations in pituitary GnRH receptor binding parameters, and the effect of castration on pituitary GnRH receptor binding were investigated in goldfish and catfish, respectively. In goldfish, GnRH receptors undergo seasonal variation with the highest pituitary content of both high and low affinity sites occurring during the late stages of gonadal recrudescence. The observed changes in pituitary GnRH receptor content correlate closely with responsiveness to a GnRH agonistin vivo in terms of serum gonadotropin (GTH) levels. In catfish, castration results in a two-fold increase in pituitary GnRH receptor content, which can be reversed by concomitant treatment with androstenedione, but not by the non-aromatizable androgen 11β-hydroxyandrostenedione; changes observed in GnRH receptor content correlate with variations in serum GTH levels and responsiveness to a GnRH agonist. In summary, the present study provides a clear evidence for seasonal variation in pituitary GnRH receptor activity in goldfish, and demonstrates a gonadal feedback mechanism regulating GnRH receptor activity in the catfish pituitary.     

日本鳗鲡产卵后生存及其繁育

为证明日本鳗鲡(Anguilla japonica)生活史最后一步一产后鳗的命运,本研究模拟产后的日本鳗鲡继续在海水中养殖,观察其存活率及繁育情况.结果表明,产后鳗在海水中停食约18 d后,体能得到恢复,部分亲鱼开始出现摄食,1个月左右全部恢复摄食,经244 d养殖,雌、雄鳗体质量增加,存活率达94.6%.随后,给产后鳗注射外源性促性腺激素(鲤鱼脑垂体匀浆CPE和人绒毛膜促性腺激素HCG)后激发其退化的性腺(卵巢和精巢)重新发育(与当年银鳗作对照).通过性腺组织切片观察产后鳗和对照鳗性腺发育成熟的全过程及其差异,发现产后鳗起初性腺发育比当年银鳗差,但经多次注射激素后,产后鳗性腺成熟与当年银鳗同步,证明产后鳗生殖细胞对激素的敏感件高.应用17α,20β-双羟孕酮和促性腺激素释放激素类似物(GnRH-A3)使催熟的产后鳗和对照鳗均产卵和排精,并孵化出仔鱼,从而有力地证明,鳗鲡产后虽体质弱,但待体能恢复后能够继续生存和繁殖.本研究旨在探讨利用产后鳗作为今后鳗鲡人工繁殖亲鳗的可行性.     

大鳍鳠脑垂体和血清生长激素水平的季节变化

根据大鳍脑垂体匀浆和血清样品的稀释曲线与鲤生长激素（ｃＧＨ）标准曲线的平行性,采用鲤生长激素的标准品和抗血清（ＲＡＧ）测定了周年中几个不同时期大鳍脑垂体和血清样品的生长激素（ＧＨ）含量,发现脑垂体和血清中的ＧＨ含量均表现出明显的季节变化。脑垂体的ＧＨ含量分别在３月份和８月份出现两个峰。４～７月的繁殖期和１１～１月的越冬期间,脑垂体的ＧＨ含量很低,而且波动不大。受水温和光周期的影响,大鳍血清ＧＨ水平表现为从冬季（１１～１月）到春季（２～４月）逐渐上升,夏季急剧升高,到夏末（７月底）达到最高,一直持续到秋季。大鳍血清ＧＨ含量的变化与生殖周期密切相关,最低的ＧＨ含量出现在性腺静止期,其次为性腺发育期,再次为性腺成熟期,在产卵期急剧升高,最高为性腺退化期。     

Effect of aromatable androgen (17‐methyltestosterone) on induced maturation of silver European eels (Anguilla Anguilla): Oocyte performance and synchronization

The reproductive performances of silver European eel in term of gonad development and egg production, employing slow‐release implants with the androgen 17‐MT (1 mg) in combination with traditional weekly injection of carp pituitary extract (CPE) was evaluated. Wild female European eels (Anguilla anguilla) underwent a standard induction protocol with CPE and were randomly divided into three groups (N‐group, no implant; Y‐group, with implant; and control, C‐group, no treatment). The results showed that 17‐MT‐treated females (Y‐group) reproduced spontaneously about 6 weeks earlier than the N‐group females with a saving of almost 40% in CPE and time of induction. Concerning artificial induction of maturation in female silver eels, our study demonstrated that they positively respond to androgen exposure also in terms of eggs productivity. Indeed, Y‐group was more productive than N‐group: in Y‐group, 11 eels ensured an eggs production that exceeded 50% of initial body weight (BW), whereas in N‐group only three eels have exceeded this value. The results suggest that 17‐MT should be considered in future protocols for the improvement of the artificial reproduction of female silver European eels.     

The GnRH systems in the brain and pituitary of normal and hCG treated European silver eels

The distribution of immunoreactive GnRH was studied in the brain and pituitary gland of normal and human chorionic gonadotrophin (hCG) injected silver eels. It was found that the general organization of GnRH systems in this species is similar to that reported in other teleosts. Cell bodies were present in the olfactory bulbs, ventral telencephalon, periventricular hypothalamus and dorsal tegmentum. No positive perikarya could be detected in the preoptic region. Only scarce fibers were observed in the proximal neurohypophysis. Treatment with hCG does not modify the distribution of GnRH but it increases the density of positive structures, in particular at the level of the pituitary. The results are discussed in relation with the present status of knowledge of the mechanisms underlying the blockage of sexual maturation in the European eel at the silver stage.     

Estimation of gonadal status of Japanese female eel <Emphasis Type="Italic">Anguilla japonica</Emphasis> by a single injection of salmon pituitary extract

ABSTRACT:   Cultivated eels often either lack a part of the ovary (abnormal) or do not have any gonadal tissue (completely sterile). These abnormalities may cause a failure of induction of sexual maturation of female eels. In order to effectively induce sexual maturation and obtain good quality eggs, female eels having normal ovaries should be selected before induction of maturation. The present study examined effects of injection of salmon pituitary extract (SPE) on serum estradiol-17β levels to estimate gonadal status of sexually immature Japanese eels. At the initial stage (just prior to SPE injection), serum estradiol-17β levels were low and no significant difference was observed among eels possessing the different gonad types (normal, abnormal, and sterile), suggesting that gonadal status cannot be estimated by the measurement of serum estradiol-17β levels. A single injection of SPE significantly increased serum estradiol-17β levels in eels possessing normal and abnormal ovaries, but not in sterile eels. Serum estradiol-17β levels significantly increased from 24 h and were high on day 3. These data indicate that eels possessing ovaries can be distinguished from sterile eels by measurement of serum estradiol-17β between 1 and 3 days after a single injection of SPE.     

The duality of teleost gonadotropins

The duality of salmon gonadotropins has been proved by biochemical, biological, and immunological characterization of two chemically distinc gonadotropins. GTH I and GTH II were equipotent in stimulating estradiol production, whereas GTH II appears to be more potent in stimulating maturational steroid synthesis. The ratio of plasma levels and pituitary contents of GTHs and the secretory control by a GnRH suggest that GTH I is the predominant GTH during vitellogenesis and early stages of spermatogenesis in salmonids, whereas GTH II is predominant at the time of spermiation and ovulation. GTH I and GTH II are found in distinctly separate cells. In trout, GTH I is expressed first in ontogeny, whereas GTH II cells appear coincident with the onset of spermatogenesis and vitellogenesis, and increase dramatically at the time of final reproductive maturation. Comparison of the amino acid sequences of polypeptides and the base sequences of cDNA revealed that salmon GTH I β is more similar to bovine FSHβ than bovine LHβ and salmon GTH II β shows higher homology to bovine LHβ than to bovine FSHβ. The existence of two pituitary gonadotropins in teleosts as well as tetrapods suggests that the divergence of the GTH gene took place earlier than the time of divergence of teleosts from the main line of evolution leading to tetrapods.     

Temporal progression in migratory status and sexual maturation in European silver eels during downstream migration

The onset of downstream migration of European eels is accompanied by a cessation of feeding and the start of sexual maturation which stresses the link between metabolism and sexual maturation, also suggesting an important role for exercise. Exercise has been tested with eels in swim tunnels and was found to stimulate the onset of sexual maturation. In this study, we have investigated the interplay between migration and maturation in the field during the downstream migration of female silver eels. Temporal changes in migratory status and sexual maturation among silver eels of the upstream Rhine River system over 3 months of the migration season (August, September and October) were determined in biometrical parameters, plasma 17β-estradiol and calcium levels, oocyte histology and gonadal fat levels. Furthermore, the ecological relevant parameters age as determined by otolithometry and health aspects indicated by haematocrit, haemoglobin and swim-bladder parasite load were measured. Silver eels were estimated to be 14 years old. A strong temporal progression in migratory stage was shown over the months of downstream migration. Catches probably represented a mix of reproductive migrants and feeding migrants of which the ratio increased over time. Furthermore, this study confirmed our hypothesis linking the migratory stage to early maturation as indicated by enlargement of the eyes, oocyte growth and fat deposition in the oocytes, exactly the same changes as found induced by exercise but not ruling out environmental influences. Migrants show extensive fat uptake by the oocytes, probably stimulated by the swimming exercise. In addition, at least 83% of the silver eels in this spawning run may have suffered from negative effects of swim-bladder parasites on their swimming performance.     

Controlled reproduction in Anguilla anguilla (L.): comparison between spontaneous spawning and stripping‐insemination approaches

This study aimed to compare the fertility of eggs between artificially matured female silver eels that spawned spontaneously and those that were spawned by manual stripping. The effects of the two methods of spawning on ovulation and fertilization rate were also investigated. For this purpose, 18 wild female European eels captured in Bonello lagoon (North Adriatic Sea) were carp pituitary extract‐injected to undergo sexual maturation and ovulation; a final injection of 17,20β‐dihydroxy‐4‐pregnen‐3‐one (DHP) was administered when at least 30% of the oocytes were fully transparent. After the DHP‐injection, nine eels were transferred to a new closed recirculating aquaculture system, where they were housed with spermiating males (sex ratio 4/1) to allow spontaneous spawning (SPT‐group); the remaining nine eels were transferred to a 250 L tank and ovulation was checked at four‐hourly intervals by manual stripping (STR‐group). The number of eggs per female in the SPT‐group was significantly greater than that in the STR‐group. Furthermore, fertilization rates in the SPT‐group were notably higher than those observed in the STR‐group. Significantly, the best performances were obtained among eels in which at least 50% of oocytes were fully transparent at the time DHP was administered. We conclude that the fertility of eggs from spontaneously spawning eels is superior to that of eggs acquired by strip‐spawning and artificial fertilization.     

Neuropeptide Y (NPY) modulatesin vitro gonadotropin in release from rainbow trout pituitary glands

This work investigated the action of neuropeptide Y (NPY) on thein vitro pituitary release of the maturing gonadotropic hormone (GtH) of the rainbow trout using a perifusion system employing trout balanced salt solution (pH 7.5) at 15°C and a 12.5 ml/h flow rate. In vitellogenic females a 20 minutes NPY application (10⁻⁷ M) induced a 20–30% decrease in GtH secretion. Removal of NPY was followed by a rebound in GTH secretion. On the contrary, in ovulated females, NPY (15 minutes, 10⁻⁷ M) directly stimulated GTH secretion. The greatest stimulation was obtained the day of ovulation where the stimulatory effect of NPY was similar to those induced by s.GnRH in the same conditions, reaching 400% of the basal GTH level. In vitellogenic females treated with 1-4-6 androstadien 3–7 dione, an inhibitor of aromatase activity, the pituitary response to NPY was similar to that obtained in ovulated females. Thus thein vitro action of NPY might depend on thein vivo steroidogenic environment.     

Gonadotropin-releasing hormone and gonadotropin in goldfish and masu salmon

Reproductive activities in vertebrates are regulated by an endocrine system, consisting of the brain-pituitary-gonad axis. In teleosts, gonadotropin-releasing hormone (GnRH) in the brain stimulates gonadotropin (GTH) release in the pituitary gland, but because of lack of the portal vessel, it is not known when and how much GnRH is released for the regulation of GTH release. There are multiple molecular types of GnRH in teleosts and several distinct populations of GnRH neurons in the brain. However, we do not know which types and populations of GnRH neurons regulate reproductive activities. Here we summarize our recent studies on GnRH and GTH in masu salmon Oncorhynchus masou and goldfish Carassius auratus. Immunocytochemistry showed the location and molecular types of GnRH neurons. Salmon (sGnRH) and chicken-II GnRH (cGnRH-II) neuronal fibers were widely distributed in the brain of both masu salmon and goldfish. Only sGnRH fibers were observed in the pituitary of masu salmon, whereas both sGnRH and cGnRH-II fibers were observed in the goldfish pituitary, indicating that species specific GnRH profiles are involved in the regulation of pituitary function in teleosts. A series of experiments in masu salmon and goldfish suggest that among GnRH neuron populations GnRH neurons in the ventral telencephalon and the hypothalamus regulate GTH release, and that GnRH of the terminal nerve origin is not essential to gonadal maturation and ovulation. The biological function of other GnRH neurons remains unkown. Two GTHs appear to be characteristic of teleost; however, regulation of reproduction by these GTHs is a question that remains to be elucidated. In salmonid species, it is proposed that GTH I stimulates early gonadal development, whereas GTH II acts in later stages. When GTH expression was examined in goldfish, both GTH I and II mRNA levels in the pituitary gland showed increases in accordance with gonadal development, unlike the sequential expression of GTH subunits in salmonids. The expression of these GTH subunit mRNAs were affected by water temperature, starvation, and steroid hormones in goldfish, but in what manner these two GTHs regulate gonadal development remains to be clarified.     

Fluctuations in gonadotropin and ovarian steroids during the annual cycle and spawning of the common carp

The main objective of the paper is to describe the annual changes in hormones associated with reproduction in the female carp under the conditions prevailing in the Israeli fish culture. Fish were sampled monthly throughout 1984; gonadosomatic index (GSI) was calculated and the diameter of ovarian follicles was measured. Gonadotropin (GTH) content in the pituitary and the circulating GTH, estradiol, testosterone and 17, 20-dihydroxy-4-pregnen-3-one (17, 20-P) were determined by specific radioimmunoassays. GTH, estradiol and testosterone showed a bimodal annual pattern. The late summer peak was associated with initial vitellogenesis while the peak in spring occurred just before spawning, which took place in April-May. A resting phase in ovarian activity was noted in June and July. The levels of 17, 20-P were very low compared with those occurring during spawning induction. The paper summarizes a previous study by our laboratory on the changes in circulating hormones, as related to oocyte stages, in female carp induced to spawn by a GTH-calibrated pituitary extract. This study associates the short but prominent peak in 17, 20-P with the presence of follicles with maturing oocytes in the ovary. A correlation was found between the percentage of oocytes with eccentric germinal vesicle initially present in ovarian biopsies of females carp and their spawning success after hypophysation. The paper describes simple means to ensure successful induction of spawning in carp by utilizing a calibrated pituitary extract and prior selection of females that would respond to the induction treatment.     

Lipid content and fatty acid composition of muscle,liver, ovary and eggs of captive-reared and wild silver Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis> during artificial maturation

ABSTRACT:   Changes in lipid content and fatty acid composition of muscle, liver and ovary of captive-reared and wild silver Japanese eel Anguilla japonica were examined during artificial maturation induced by salmon pituitary homogenate (SPH) injections. Although the relative levels of n-3 and n-6 highly unsaturated fatty acids (HUFA) in liver and ovary were higher than in muscle in both captive and wild silver eels before SPH injection, these tended to decrease with maturation. The relative levels of n-6 HUFA in muscle, liver, ovary and eggs of wild silver eels were remarkably higher than those in captive eels. Therefore, we attempted to alter the ratio of n-6 HUFA in eggs by feeding eels a diet supplemented with linoleic acid-rich plant oil. Although the percentage of n-6 polyunsaturated fatty acids to total fatty acids in eggs of eels fed the supplemented diet was similar to wild silver eels, the percentage of n-6 HUFA remained remarkably lower than in wild silver eels. Hence, it appears that the supplemented diet affected the fatty acid composition of eggs, but did not result in much conversion of linoleic acid to its higher homologs in eels.     

Hypothalamic peptides influencing growth hormone secretion in the goldfish,Carassius auratus

In vivo andin vitro techniques were used to examine the influence of various vertebrate peptides on growth hormone (GH) secretion in the goldfish. Tetradecapeptide somatostatin (SRIF-14) was found to inhibit GH secretionin vitro from perifused pituitary fragments, whereas similar concentrations of a salmonid SRIF peptide (sSRIF-25) did not affect GH secretion from the goldfish pituitary fragments. This indicates that SRIF receptors on the goldfish pituitary are very specific for SRIF-14-like peptides. Salmon gonadotropin (GTH)-releasing hormone (sGnRH) was found to elevate serum GH levels in male goldfish. The dopamine antagonist pimozide alone or injected in combination with sGnRH did not influence serum GH levels, although injection of pimozide alone significantly elevated serum GTH levels, in addition to potentiating the effects of sGnRH on GTH secretion. sGnRH stimulated GH secretion from goldfish pituitary fragmentsin vitro, indicating that sGnRH acts directly at the level of the pituitary to stimulate GH secretion in the goldfish. These results suggest that GnRH may also function as a GH-releasing factor in the goldfish, although the release-inhibitory factors for GH and GTH secretion do appear to be separate and distinct. Two human GH-releasing hormone (hGHRH) peptides were found to be ineffective in altering GH secretionin vitro from the perifused pituitary fragments. Consequently, a role for a mammalian GHRH-like peptide in the hypothalamic regulation of GH secretion in the goldfish remains questionable.     

Extrapituitary gonadotropin-releasing hormone (GnRH) binding sites in goldfish

In teleosts, as in other vertebrates, the secretion of pituitary gonadotropin (GTH) is mediated by the hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH). Recent findings in teleosts indicate that GnRH receptors are not restricted to the pituitary gonadotropes and are also associated with somatotropes as well as being present in a number of other tissues. In the present study, we provide novel information on GnRH binding in a number of extrapituitary tissues in goldfish. However, we do not intend to provide full characterization of GnRH binding sites in various extrapituitary tissues in goldfish as this would clearly be outside the scope of this paper. In this study we examined GnRH binding in a number of extrapituitary tissues in goldfish and observed specific binding in ovary, testis, brain, liver and kidney. No specific GnRH binding was observed in muscle, skin, gut, gill and heart. In general, the present findings together with the results of other studies carried out in our laboratory demonstrate that mature goldfish ovary and testis contain two classes of GnRH binding sites, high affinity/low capacity and low affinity/high capacity sites with binding characteristics similar to those of the pituitary GnRH receptors. The brain of goldfish was also found to contain two classes of GnRH binding sites, a super-high affinity/low capacity and a low affinity/high capacity sites. Furthermore, study of goldfish liver and kidney demonstrated the presence of a single class of GnRH binding sites with characteristics different from those of pituitary, ovary, testis and brain. Overall, it is evident that goldfish contains a family of GnRH binding sites which can be classified into four groups based on binding affinities: 1) A class of high affinity binding sites present in the pituitary, ovary and testis, 2) a class of super high affinity sites so far only detected in the brain, 3) a class of intermediate-affinity GnRH binding sites in the liver and kidney, and 4) a class of low affinity binding sites present in all the tissues containing specific GnRH binding sites except for liver and kidney.     

鲤鱼血清中促性腺激素、17β雌二醇含量的周年变化

应用放射免疫测定法,对雌、雄鲤血清中促性腺激素(GTH)和17β-雌二醇(17β-E_2)的含量进行周年测定。GTH的常年分泌量,雌鲤平均为8.94±5.94ng/ml(12月份含量最低,峰值在1月份);雄鲤常年平均为8.72±5.20ng/ml(10月份含量最低,峰值在11月份)。17β-E_2的含量,雌鲤1-4月份较高,平均为2019.48±754.49pg/ml(全年峰值在2月份)。产卵季节后,卵巢开始退化,17β-E_2含量下降。5—8月平均为454.58pg/ml,产卵前后差异性显著。这表明性腺的发育和成熟与血清中17β-E_2含量变化的密切相关性以及雌二醇对GTH分泌的反馈抑制作用。     

Gonadotropin-releasing hormones in the African catfish: molecular forms, localization, potency and receptors

Two gonadotropin releasing hormones (GnRHs) were identified in the African catfish: chicken GnRH-II (cGnRH-II) and catfish GnRH (cfGnRH). Immunological screening of HPLC fractions from pituitary extracts indicated a third GnRH which co-eluted with lamprey GnRH-III. However, mass determination and amino acid sequencing identified this material as isotocin. This underlines the risk of identifying multiple forms of GnRH in tissue extracts on the basis of immunoreactivity in HPLC fractions. In vivo and in vitro studies demonstrated that cGnRH-II is an over 100-fold more potent gonadotropin (GTH) secretagogue than cfGnRH. This correlates with the respective receptor affinities. The presence of both GnRHs in the pituitary gland suggests that they may modulate each other's GTH release activity. Sub-threshold or low doses of cGnRH-II partly inhibited cfGnRH-induced GTH II secretion. Conversely, combinations of sub-threshold or low doses of cfGnRH with effective doses of cGnRH-II led to increases in GTH II levels similar to those induced by cGnRH-II alone. Combinations of submaximally effective dose of the 2 peptides resulted in additive effects. Hence, both GnRHs participate in the regulation of GTH II release, and their relative concentrations may determine the overall effect. Immunocytochemistry, using anti-bodies against the respective recombinant GnRH associated peptides (GAPs), as well as in situ hybridization showed that cfGnRH neurones are scattered in the ventral forebrain and project into the pituitary gland, while cGnRH-II neurones are confined to the midbrain tegmentum and without projections to the pituitary gland. Transfection experiments with GnRH receptor cDNA shows ligand activation characteristics similar to those of the native GnRH-R. Autoradiographic studies and hormone release studies indicate that GnRH-Rs in the African catfish pituitary gland are restricted to the gonadotrophs.