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.     

Molecular cloning and expression of salmon pituitary hormones

A cDNA library was prepared from chinook salmon pituitaries. Growth hormone (GH), prolactin (PRL) and the β subunit of gonadotropin (GTH) genes were screened using synthetic oligonucleotides as probes. Full size cDNA clones coding for these polypeptide hormones were isolated and characterized. The cDNA sequences for PRL and βGTH have been reported earlier from our laboratories. The cDNA clone for GH contains 1148 bp and codes for a preGH of 210 amino acids. The chinook salmon GH, reported in the present investigation, differs from chum salmon GH in only 1 amino acid, and from coho salmon GH in 5 amino acids. Plasmids containing modified nucleotide sequences coding for GH, PRL and βGTH were constructed individually into an expression vector using the heat-inducible λ pL promotor. Mature PRL, GH and unglycosylated βGTH were expressed in the bacteria at elevated temperature.     

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.     

Changes in plasma somatolactin levels during spawning migration of chum salmon (Oncorhynchus keta)

Plasma somatolactin (SL) concentrations were examined in chum salmon in relation to gonadal maturation; immature salmon in the Bering Sea at various stages of maturation, and mature salmon during upstream migration caught at the ocean, bay and river. Plasma SL concentrations as well as plasma prolactin (PRL) and growth hormone (GH) levels in the immature fish caught in the Bering Sea were maintained essentially at similar levels. Plasma SL in mature salmon increased significantly from the fish in the ocean to the fish in the river in both sexes. Although all the fish had fully developed gonads, females completed ovulation while still in the bay, whereas final spermeation in males was achieved after entry into the river. Thus, no clear correlation was seen between plasma SL levels and final gonadal maturation. On the other hand, plasma PRL concentrations in both male and female fish were higher in the fish in the river than those in the ocean and bay, and plasma GH levels were higher in both sexes in the fish in the bay and river than those in the ocean. Plasma levels of triglycerides, glucose, free fatty acids and ionized sodium and calcium were also examined. Significant-negative correlations were seen between plasma SL and plasma ionized calcium in mature male salmon, and between plasma SL and plasma triglycerides in mature female salmon. Although our findings do not rule out the possibility of the involvement of SL in final maturation, the results indicate that SL seems to be involved at least in energy and/or calcium metabolism during the spawning migration.     

Immunohistochemical investigation of the pituitary of the sturgeon (Acipenser baeri,Chondrostei)

An immunohistochemical study of the sturgeon (Acipenser baeri) pituitary was undertaken using antisera directed against hormones from various classes of vertebrates, including the only pituitary hormone available from sturgeon, gonadotrophin. A positive reaction was obtained after application of antisera towards the following hormones 1–24 synthetic ACTH (1-24 ACTH), melanophore stimulating hormone (MSH), ovine prolactin (oPRL), ovine growth hormone (oGH), salmon growth hormone (sGH), carp gonadotrophin (cGTH) and its beta subunit (cGTH), sturgeon gonadotrophin (aciGTH), carp thyrotrophin (cTSH) and subunit of the human thyrotrophin (hTSH). The results demonstrate that, in general, the sturgeon pituitary resembles that of teleosts as regards the distribution of the different cell types: ACTH and PRL cells in the rostral pars distalis, GTH, TSH and GH cells in the proximal pars distalis and MSH and PAS-cells in pars intermedia. In addition to the topographical organization of the sturgeon pituitary, this study provides data on the immunological relationships between sturgeon pituitary hormones and those of other vertebrates.     

Effects of silvering state on induced maturation and spawning in wild female Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis>

ABSTRACT:   The effects of silvering state of wild female Japanese eels Anguilla japonica on the success of induced maturation and the following spawning were examined. Thirty-eight females, collected in Mikawa Bay, were divided into four stages based on their silvering state: yellow (Y1), late-yellow (Y2), silver (S1) and late silver eels (S2). Despite injections of salmon pituitary extract (SPE) through the standard technique, Y1 and Y2 eels did not respond to the treatment with undeveloped gonad (gonad-somatic index [GSI]: 0.3–0.9), and all these females died by 5 weeks, probably due to an abnormal physiological condition. Most S1 (81%) and S2 eels (100%) matured completely (GSI: 17.8–51.4), and finally spawned successfully (69% for S1, 89% for S2). S2 eels fully matured with oocytes of over 750 μm in diameter by significantly smaller number of injections of SPE (5–6 times) than the case of S1 eels (6–8 times). The amount of eggs released by S2 eels (0.65 ± 0.11 g/fish per body weight [BW]) was significantly larger than those by S1 eels (0.54 ± 0.09 g/fish per BW). There was no difference in fertilization and hatching rates between eggs released by S1 eels and those of S2 eels. These results indicate that the success of induced maturation and spawning in wild female Japanese eels depends on their silvering state, and matured eggs can be obtained efficiently through the use of S2 eels rather than other stages.     

Effects of 11-ketotestosterone and gonadotropin-releasing hormone on follicle-stimulating hormone and luteinizing hormone gene expression in castrated and sham-operated male red seabream <Emphasis Type="Italic">Pagrus major</Emphasis>

ABSTRACT:   In order to clarify the roles of androgen and gonadotropin-releasing hormone (GnRH) on gonadotropin (GTH; luteinizing hormone [LH] and follicle stimulating hormone [FSH]) synthesis, effects of castration and implantation of GnRH analog (GnRHa) or 11-ketotestosterone (11-KT) on expression of GTH subunit, α-glycoprotein subunit (αGSU), FSHβ, and LHβ genes, during the early spermatogenic stage in male red seabream Pagrus major were examined. Male red seabream underwent castration or sham-operation and were subsequently implanted with cholesterol pellets containing GnRHa, silicone capsules filled with 11-KT, or blank capsules (control). FSHβ mRNA levels increased due to castration, and it was reversed by treatment with 11-KT. 11-ketotestosterone treatment also decreased FSHβ mRNA levels in sham-operated fish. These results suggest that 11-KT acts on the pituitary to suppress FSH synthesis in male red seabream. On the other hand, neither castration nor replacement of 11-KT in castrated fish had effects on LHβ mRNA levels, whereas 11-KT treatment had slightly but significantly decreased LHβ mRNA in sham-operated fish. αGSU mRNA levels were not changed by castration or 11-KT treatment in both sham-operated and castrated fish. Meanwhile, treatment with GnRHa significantly decreased FSHβ mRNA levels in sham-operated fish, but not in castrated fish. This suggests that GnRHa may down-regulate expression of FSHβ mRNA through the production of 11-KT in testis. LHβ and αGSU mRNA levels in sham-operated fish, but not in castrated fish, were significantly elevated by treatment with GnRHa.     

Long-term starvation in the European eel: general effects and responses of pituitary growth hormone-(GH) and somatolactin-(SL) secreting cells

European eels reaching the silver stage stop feeding in freshwater and during their spawning migration to the Sargasso sea (6000 km at least in the Ocean). The total duration of this exceptional fast is not well known. Few data are available on the general condition and the endocrine responses to starvation of migrating eels. In this study male (silver) and female (yellow and silver) eels were kept in freshwater without food for various lengths of time. Animals were killed after 7 months to 3 or 4 years. After a gradual decrease, the final body weight was reduced by 84% in males (52 months) and 67–69% in females (up to 4 years). The condition factor (K) followed a parallel curve. In the pituitary gland, GH cells were hypertrophied and highly stimulated. Their cross-sectional area was negatively correlated to K. Large GH cells remained well immunostained with an anti-eel GH serum after 7 to 12 months of starvation. In the leanest eels, the immunostaining was often reduced and many GH cells appeared degranulated, suggesting a low hormonal storage. In contrast, SL cells were reduced in size and number in the anterior half of the neurointermediate lobe (NIL), but showed a more heterogeneous picture in the caudal portion. GH and SL cell activities seemed to be negatively correlated in starved fish and controlled by different mechanisms. GH appears to play a major role in the long-term survival of fasted eels; SL does not seem to be involved.     

Immunoreactive changes in pituitary FSH and LH cells during seasonal reproductive and spawning cycles of female chub mackerel <Emphasis Type="Italic">Scomber japonicus</Emphasis>

The physiological functions of pituitary gonadotropins (GtHs) are well established in higher vertebrates, whereas those in teleosts are still poorly understood. To describe the role of GtHs during gonadal development of female chub mackerel Scomber japonicus, changes in follicle-stimulating hormone (FSH) and luteinizing hormone (LH) cells were investigated immunohistochemically during the seasonal reproductive and spawning cycles. FSH and LH cells were identified in the different cell types of the proximal pars distalis (PPD); FSH cells were located in the central PPD, whereas LH cells were localized along the border of the pars intermedia. To examine changes in FSH and LH cells, the percentage of FSH or LH cell-occupying area in the PPD was evaluated and represented as FSHβ-immunoreactive (ir) or LHβ-ir levels, respectively. FSHβ-ir levels increased significantly from immature to the completion of vitellogenesis, whereas LHβ-ir levels were maintained at high levels from early vitellogenesis to post-spawning. During the spawning cycle, which consisted of four stages from just after spawning to the next oocyte maturation, both FSHβ-ir and LHβ-ir levels showed no significant changes among different stages; however, LHβ-ir levels remained relatively high, and FSHβ-ir levels were constantly low. These results suggest that both FSH and LH may be involved in vitellogenesis and LH may act at final oocyte maturation in female chub mackerel, although the role of FSH during the spawning cycle is still unclear.     

Plasma somatolactin levels in coho salmon (Oncorhynchus kisutch) during smoltification and sexual maturation

Somatolactin (SL) is a novel pituitary hormone recently characterized in several fish species. Structural analyses have shown that SL belongs to the growth hormone/prolactin family, and that it is a highly conserved protein. SL is synthesized by the periodic acid/Schiff-positive cells in the pars intermedia, but has an as yet unidentified function(s). We have recently developed a homologous radioimmunoassay for coho salmon SL and measured plasma levels of SL during two stages of the coho salmon life cycle, smoltification and sexual maturation. During smoltification, plasma levels of SL changed almost in parallel with plasma levels of thyroxine; levels increased as morphological indices of smoltification appeared and decreased as smoltification was completed. Following this period, SL levels remained low until the spring prior to spawning. In a separate study, plasma levels of SL were measured in sexually maturing coho salmon that remained in fresh water throughout their life cycle. During the year of sexual maturation, plasma levels of SL gradually increased from the spring onward, reaching peak levels at the time of spawning in November and December. These data are similar to those previously reported for sexually maturing coho salmon that were maintained in seawater prior to spawning (Rand-Weaver et al. 1992). Therefore, increases in plasma SL levels occurred in sexually maturing fish irrespective of whether they were maintained in fresh water or seawater. Peak levels at spawning were higher than those observed during smoltification. Possible roles for SL in metabolism and reproduction are discussed.     

Close association of gonadotropin-releasing hormone fibers and gonadotropin, growth hormone, somatolactin and prolactin expressing cells in pejerrey, Odontesthes bonariensis

The purpose of this study was to determine if there is any association between immunoreactive (ir) gonadotropin-releasing hormone (GnRH) fibers with different pituitary endocrine cell types in the pejerrey, Odontesthes bonariensis. Using a monoclonal antibody raised against mammalian GnRH (mGnRH) (LRH13), ir-GnRH fibers were observed passing through the pituitary stalk and reaching the three areas of the pituitary gland: rostral (RPD) and proximal pars distalis (PPD) and pars intermedia (PI). Double labeled immunocytochemistry showed ir-GnRH fibers in close association with prolactin (PRL)-producing cells in the RPD, growth hormone (GH)-producing cells in the PPD, gonadotropin (GtH)-producing cells in the PPD and the external border of the PI, and with somatolactin (SL)-producing cells in the PI. Our results show, direct morphological evidences of a close association of GnRH fibers with GH, PRL, GtH and SL-expressing cells. These results would suggest that GnRH has a broad role in the regulation of the secretion of different pituitary hormones.     

Changes in Serum Steroid Hormones and Vitellogenin Levels in Cultured Female European Eels Anguilla anguilla during Artificially Induced Ovarian Development

Changes in serum concentrations of estradiol-17β, testosterone, 17α,20βdihydroxy-4-pregnen-3-one, and vitellogenin were investigated during ovarian development induced by injection of a salmon pituitary extract in cultured European eel Anguilla anguilla . Vitellogenesis was induced with a weekly dose of 50 mg pituitary extract/kg body weight. In eels receiving that dose, gonadsomatic indices ranged from 20–43% after the 10th–11th weekly injection. Body weights were relatively stable during vitellogenesis, but increased dramatically during final maturation. Serum estradiol-17β levels increased slightly during vitellogenesis and peaked at an average of 6.95 ng/mL at final maturation. The profile of serum vitellogenin followed that of estradiol-17β which increased markedly from an average of 0.36 to 20.72 mg/mL. Control levels of vitellogenin were undetectable throughout the study. Average serum levels of testosterone increased to a peak of 17.74 ng/mL in the early stage of vitellogenesis, followed by a sharp drop to initial levels (3.86 ng/mL) in the late stage of vitellogenesis, and then increased again to an average of 8.84 ng/mL at the final maturation stage. Serum 17α,20β-dihydroxy-4-pregnen-3-one was not detected (<50 pg/mL) throughout the experiment. Profiles of serum estradiol-17β, testosterone, and vitellogenin observed during ovarian development appear different from those found in salmonids and other teleosts. This study indicates, however, that cultured European eels are a useful model for study of gonadal maturation in the eel.     

Effects of temperature on vitellogenesis in Japanese eel Anguilla japonica

ABSTRACT:   Cultured immature female Japanese eels acclimated to sea water at either 10 or 20°C were treated weekly with salmon gonadotropin fraction (sGTH) in order to investigate the effects of water temperature on artificial induction of ovarian maturation. In eels maintained at 20°C, ovulation was induced in 11 of 18 fish during the experimental period of 13 weeks, whereas at 10°C all fish showed a low gonadosomatic index (GSI) at the end of the experiment. Plasma vitellogenin levels were higher in eels kept at 20°C than in eels at 10°C throughout the experiment. However, no significant differences were observed in the plasma testosterone and estradiol-17β levels between groups. Eels pretreated with sGTHs at 10°C for 13 weeks were separated to two different temperature (10 and 20°C) groups, and received the same weekly sGTH injections. In eels transferred to 20°C, plasma vitellogenin levels, GSI and oocyte diameter were increased, but these values were maintained at low levels in eels that remained at 10°C. These results clearly indicate that water temperature is an important factor regulating vitellogenesis in the Japanese eel.     

Molecular characterization,tissue distribution of Follicle‐Stimulating Hormone (FSH) beta subunit and effect of kisspeptin‐10 on reproductive hormonal profile of Catla catla (Hamilton, 1822)

Gonadotropin (GTH) hormones are glycoprotein which stimulates gonadal maturation in vertebrates. Follicle stimulating hormone is involved in initiation of gametogenesis and regulation of gonadal growth. FSHβ has been cloned and characterized from the brain of Catla catla. The FSHβ full‐length of cDNA sequence of 523 bp comprised 3, 394 and 128 bp of 5′‐UTR, open reading frame (ORF) 3′‐UTR respectively. The coding region of C. catla FSHβ encoded a peptide of 130 amino acids. Phylogenetic analysis of C. catla FSHβ deduced amino acid sequence showed high similarity with Gobiocypris rarus followed by goldfish, Carassius auratus. The qPCR result shows that FSHβ mRNA is mainly expressed in pituitary while moderate and low expression was observed in testis and ovary respectively. Chitosan‐nanoconjugated kisspeptin‐10 (CK‐10) of particle size 125 nm, polydispersity index of 0.335 to 0.65 and zeta potential of ?34.95 mV were synthesized and evaluated at against naked kisspeptin‐10 for their reproductive hormonal profile. Treatment of fish with CK‐10 showed controlled and sustained surge of the reproductive hormones (FSH & LH) with peak at 12 h. The hormone levels of naked kisspeptin‐10 treated fish decline after 6 h. The sustained release of this CK‐10 will help in reducing maturation age, synchronization of ovulation and spawning in fish. This is the first report on use of chitosan‐nanoconjugated kisspeptin‐10 (CK‐10) for reproduction in fish.     

Immunohistochemical localization of three GnRH systems in brain and pituitary of Japanese flounder

ABSTRACT:   To clarify the possible roles of gonadotropin-releasing hormone (GnRH) in the reproduction of Japanese flounder Paralichthys olivaceus , localization of salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and sea bream GnRH (sbGnRH) immunoreactive (ir) cell bodies and fibers in the brain and pituitary were examined together with follicle stimulating hormone (FSH) and luteinizing hormone (LH)-ir cells in the pituitary by immunohistochemistry. sGnRH-ir cell bodies were localized in the ventromedial part of the rostral olfactory bulb and cGnRH-II-ir cell bodies were restricted to the midbrain tegmentum, while sbGnRH-ir cell bodies were evident in the preoptic area. sGnRH-ir fibers were distributed throughout the brain, especially abundant in the forebrain. cGnRH-II-ir fibers were also scattered in many areas of the brain with abundance in the midbrain, but sbGnRH-ir fibers were observed in the preoptic–hypothalamic area and innervated the pituitary. In the pituitary, neither sGnRH-ir fibers nor cGnRH-II-ir fibers were found, but sbGnRH-ir fibers were profuse in the neurohypophysis and invaded the proximal pars distalis, targeting FSH and LH cells. These results suggest that three GnRH systems can play different physiological roles in the brain of Japanese flounder. Among them, sbGnRH is considered to be involved in reproduction by stimulating gonadotropin secretion, while sGnRH and cGnRH-II can function as a neurotransmitter and/or neuromodulator within the brain in this species.