Salmon Pituitary Homogenate Research Articles

Pretreatment Reproductive Stage and Oocyte Development Induced by Salmon Pituitary Homogenate in the Japanese Eel <i>Anguilla japonica</i>

Pretreatment Reproductive Stage and Oocyte Development Induced by Salmon Pituitary Homogenate in the Japanese Eel <i>Anguilla japonica</i>

Changes in transcription of pituitary glycoprotein hormone α and gonadotropin IIβ subunits during ovarian development induced by repeated injections of salmon pituitary homogenate in the Japanese eel, Anguilla japonica

To investigate transcription of pituitary glycoprotein hormone a and gonadotropin IIβ (GTH IIβ) subunits in female Japanese eel, gene quantitation systems using reverse transcription-polymerase chain reaction (RT-PCR) were developed, using β-actin as an internal control gene. These systems were not only sensitive, but also precise, with low coefficients of variation within and between assays (below 20 and 30%, respectively). Using these quantitation systems, we investigated how transcription of both subunits changed in Japanese eel during ovarian development induced by repeated injections of salmon pituitary homogenate (SPH). Transcriptional level of pituitary glycoprotein hormone α was similar to that of s-actin before SPH injection, and increased slowly during ovarian development, reaching a maximum (3-fold increase over initial levels) at the migratory nucleus stage. In contrast, the level of GTH IIβ mRNA was very low (about one-tenth that of β-actin) before SPH injection, but expression increased markedly during ovarian development with an approximate 120-fold increase over initial levels (12 times that of β-actin) at the migratory nucleus stage. These results suggest that GTH II synthesis in female eel was activated during ovarian development induced by repeated SPH injections. The increase in mRNA levels of both subunits mRNA is probably due to sex steroids produced in eel ovarian follicles. Unexpectedly, transcriptional level of GTH IIβ was abnormally high compared to that of pituitary glycoprotein hormone α at the migratory nucleus stage. These results suggest that repeated SPH injections induced aberrant endocrine conditions in female Japanese eels.

トラフグ養成親魚からの採卵技法の開発

We developed an effective method for the induction of oocyte maturation and ovulation in the cultured tiger puffer Takifugu rubripes by hormonal treatments, in order to supply the fertilized eggs for seedling production. Ovarian biopsy just prior to the hormonal treatments revealed the oocyte diameter and developmental stages of the oocytes.A luteinizing hormone-releasing hormone analogue (LHRH-a) cholesterol pellet (400μg/kg) implantation successfully induced oocyte maturation for three-year-old cultured tiger puffer which had yolk accumulated ovaries. Thereafter, single injection of HCG (500IU/kg) combined with chum salmon pituitary (SP) homogenate (7mg/kg) was applied to the fish to synchronize ovulation among fish. Ovulation mainly occurred between 96 and 120 hours after HCG+SP injection. Thus, a technique for obtaining eggs of high quality from the cultured tiger puffer was developed.

Hepatic Estrogen Receptors in the Japanese Eel, Anguilla japonica: Characterization and Changes in Binding Capacity during Artificially-induced Sexual Maturation

Estrogen receptors were identified in cytosolic and nuclear extracts of livers of female Japanese eel, Anguilla japonica. A single class of high affinity binding sites was found, with a Kd=0.97 nM for the cytosolic estrogen receptor (cER) and Kd=0.85 nM for the nuclear estrogen receptor (nER). Binding of both the cER and the nER was specific for estrogens (diethylstilbestrol: DES>estradiol-17β: E2>>estriol: E3>estrone: E1). These binding characteristics of ERs were quite different from those of the serum estrogen-binding component; [3H]E2 binding to serum was not saturable, and was displaced by testosterone but not by DES, E1 or E3. The relationships between the levels of hepatic ERs, circulating E2 and vitellogenin (VTG) during artificial maturation of cultivated female eels were examined, using eels injected weekly with chum salmon pituitary homogenate at a dose of 20 μg/g-body weight. Serum E2 levels were constantly low during pre- to midvitellogenesis, and dramatically increased in the migratory nucleus stage. However, VTG levels gradually increased from early to midvitellogenesis, and were greatly elevated in the migratory nucleus stage. Hepatic cER levels slightly increased in early vitellogenesis, and then increased significantly from midvitellogenesis to the migratory nucleus stage. In contrast, nER levels did not change significantly, although nER levels in the migratory nucleus stage were higher than those at other stages. The changes in cER levels represent increased hepatic responsiveness to estrogenic stimuli during artificial maturation. Lack of change in nER levels may be a feature of artificial maturation compared to sexual maturation in nature.

Changes in serum steroid hormones and steroidogenic ability of ovarian follicles during artificial maturation of cultivated Japanese eel, Anguilla japonica

Serum levels of oestradiol-17β (E 2), testosterone (T) and 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DHP) were assessed by radioimmunoassay during induction of sexual maturation of female Japanese eel ( Anguilla japonica) by chum salmon pituitary homogenate (SPH) treatment. SPH stimulated vitellogenic growth in about 50% of the eels. Serum E 2 levels in eels which responded to SPH remained low for the first 10 weeks after the start of SPH injections, and tended to increase after 12 weeks. Levels increased further after completion of vitellogenesis. Serum levels of T increased gradually as SPH treatment progressed, and remained higher than E 2 levels during the first 12 weeks. A further increase in T levels occurred after completion of vitellogenesis. 17α,20β-DHP was not detected in serum during the experimental period. In vitro production of E 2, T and 17α,20β-DHP by ovarian follicles in response to forskolin (an activator of adenylate cyclase), and changes in the ability of follicles to convert exogenous pregnenolone or 17α-hydroxyprogesterone (17α-OHP) to E 2, T and 17α,20β-DHP, and exogenous T to E 2, were examined using 18 h incubations. Forskolin had no effect on the production of any measured steroid by follicles at any stage of development. Production of E 2, T and 17α,20β-DHP in the absence of exogenous substrates was low. The ability of follicles to produce E 2 from pregnenolone, 17α-OHP or T was low during vitellogenesis, followed by an increase during the migratory nucleus stage. The ability of follicles to produce T from pregnenolone and 17α-OHP gradually increased during vitellogenesis, followed by a further increase or a slight decrease during the migratory nucleus stage. The conversion of pregnenolone or 17α-OHP to 17α,20β-DHP increased during vitellogenesis, followed by either a further increase or a decrease during the migratory nucleus stage. A good correlation existed between serum levels of E 2 and aromatase activity (the ability of follicles to convert T to E 2). E 2 production by follicles was shown to depend largely on aromatase activity, while T production by follicles appeared to be depend largely on SPH-induced pregnenolone production. Increased aromatase activity at the migratory nucleus stage may inhibit 17α,20β-DHP production and spontaneous final oocyte maturation and ovulation.

Induced breeding and larval rearing experiments with milkfish Chanos chanos (Forskal) in The Philippines

Salmon pituitary homogenate was used alone or in combination with human chorionic gonadotropin, to induce spawing in captive and wild adult milkfish at ambient temperature (26–30°C) and salinity of 34%.. Healthy or slightly injured females having oocytes with a minimum mean diameter of 0.66 mm could be induced to spawn in captivity using a total dose of 20 mg SPH/kg + 3000 IU HCG/kg given in two injections. Badly injured females did not respond readily to the hormone and required more than two injections to induce ovulation. Milkfish larvae were reared successfully to metamorphosis using only Chlorella-fed rotifers during the first 10 days. Survival rates were greatly improved when, aside from Chlorella and Chlorella-fed rotifers, Isochrysis galbana and Tetraselmis chuii were added to the rearing tanks.

Increased calcitonin levels during ovarian development in the eel, Anguilla japonica

Female silver eels ( Anguilla japonica) were matured with repeated injections of chum salmon pituitary homogenate. Blood samples and ovaries were taken at various stages of ovarian development for the analysis of serum immunoreactive calcitonin (CT), Ca content, and gonadosomatic index. Serum CT levels gradually increased in accordance with an increase in gonadosomatic index and the highest level was observed at the time of ovulation. An increase in serum Ca level was also observed during ovarian development. These results suggest an involvement of CT in sexual maturation in the eel.