Keiji Hirose

Artificial induction of maturation and fertilization in the Japanese eel, Anguilla japonica.

Repeated injections of salmon pituitary extract (20 mg per fish per week) induced vitellogenesis in feminized, cultivated Japanese eels (Anguilla japonica). Oocytes were attained at the migratory nucleus stage after 11 or 12 injections. Addition of 17,20β-dihydroxy-4-pregnen-3-one (DHP) into the incubation medium induced germinal vesicle breakdown (GVBD) in the oocytes at the migratory nucleus stage. An injection of DHP (2 µg g-1 BW), given 24h after an injection of salmon pituitary extract (20 mg fish-1), succeeded in inducing maturation and ovulation in females which contained occytes at the migratory nucleus stage. Most fish ovulated 15–18h following the DHP injection. Eggs that were ovulated within 15h after the DHP injection showed high fertility and hatchability, but eggs ovulated 18 or 21h after the DHP injection, showed considerably lower fertility and hatchability. A delay between ovulation and stripping of the eggs rapidly decreased both the fertility and hatchability within 6–9h after ovulation, indicating that artificial fertilization must be carried out immediately after ovulation. Repeated injections of human chorionic gonadotropin (hCG) at a concentration of 1 IU g-1 BW week-1 induced spermatogenesis, spermiation, and the acquisition of potential for sperm motility in cultivated males. Most males spermiated after the fifth or sixth injection of hCG, and the milt weight gradually increased and remained constant (1–2 g) from the 11th to 31th injection. Sperm motility peaked 24h after each weekly injection, and decreased from the 3rd day after the injection. Potassium ions are an essential constituent for the maintenance of motility in the eel spermatozoa. Artificial seminal plasma containing 15.2 mM KCl is applicable as a milt diluent. Using these techniques developed for female and male eels, we have succeeded in obtaining many fertilized eggs from cultivated eels.

Relative in vitro effectiveness of 17α,20β-dihydroxy-4-pregnen-3-one and other pregnene derivatives on germinal vesicle breakdown in oocytes of ayu (Plecoglossus altivelis), amago salmon (Oncorhynchus rhodurus), rainbow trout (Salmo gairdneri), and goldfish (Carassius autatus)

The relative effectiveness of several pregnene derivatives, which had previously been identified in the ovaries of ayu (Plecoglossus altivelis), on germinal vesicle breakdown (GVBD) was investigated in vitro using folliculated oocytes of four species of teleosts, ayu, amago salmon (Oncorhynchus rhodurus), rainbow trout (Salmo gairdneri), and goldfish (Carassius auratus). Although some species differences existed in the relative effectiveness of each steroid on GVBD, 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17 alpha, 20 beta-diOHprog) was consistently the most potent inducer of final oocyte maturation. Both progesterone and 17 alpha-hydroxyprogesterone were effective at relatively high concentrations. Of the 5 beta-reduced metabolites, 17 alpha, 20 beta-dihydroxy-5 beta-pregnan-3-one was almost as effective as 17 alpha, 20 beta-diOHprog in oocytes of amago salmon and rainbow trout, while the other 5 beta-reduced compounds (3 alpha-hydroxy-5 beta-pregnan-20-one, 17 alpha-hydroxy-5 beta-pregnane-3,20-dione, 3 alpha, 17 alpha-dihydroxy-5 beta-pregnan-20-one, 5 beta-pregnane-3 alpha, 17 alpha, 20 beta-triol, and 5 beta-pregnane-3 beta, 17 alpha, 20 beta-triol) were almost or totally ineffective at the concentrations tested (1-0.001 micrograms/ml). These bioassay results, together with previous findings on the capacity of the ovary to produce 17 alpha, 20 beta-diOHprog, indicate that 17 alpha, 20 beta-diOHprog is the natural maturation-inducing steroid hormone common to three species of Salmoniformes, ayu, amago salmon, and rainbow trout. These results, however, only suggest that 17 alpha, 20 beta-diOHprog is involved in maturation of goldfish oocytes, since supportive physiological and biochemical data are lacking. Possible regulatory roles of 5 beta-reduced metabolites on steroid-induced oocyte maturation are discussed.

Changes in fertilization and hatching rates with time after ovulation induced by 17, 20β-dihydroxy-4-pregnen-3-one in the Japanese eel, Anguilla japonica

Abstract This study examined the time to ovulation in artificially matured Japanese eel following an injection of 17, 20β-dihydroxy-4-pregnen-3-one (DHP), and changes in fertilization and hatching rates at various times after ovulation. Thirteen females, showing body weight indices (body weight/initial body weight × 100) of 119 ± 2.3% with oocytes at the migratory nucleus stage after 9–12 weekly injections of salmon pituitary extract (20 mg pituitary powder /fish/week), received an injection of salmon pituitary extract (20 mg/fish) followed 24 h later by a DHP injection (2μg/g BW) for induction of ovulation. Eleven of 13 females ovulated within 23 h of the final injection; two, seven, and two females ovulated at 14–17 h, 17–20 h and 20–23 h after the final injection, respectively. The remaining two did not ovulate within 26 h of the injection. Eggs from three out of the 11 ovulated females showed relatively high percent fertility (89.6-39.5%) and hatching rates (47.6-15.3%) when they were fertilized after the first detection of ovulation. Both percent fertilization and hatching rates of eggs retained in the body cavity decreased rapidly within 6–9 h after ovulation. These results indicate that ovulation induced by DHP treatment occurs after a short but well-defined period of time, and that artificial fertilization must be carried out immediately after ovulation in order to obtain good quality eggs.

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.

In vitro metabolism of 4-pregnenes in ovaries of a freshwater teleost, the ayu (Plecoglossus altivelis): Production of 17α,20β-dihydroxy-4-pregnen-3-one and its 5β-reduced metabolites, and activation of 3β- and 20β-hydroxysteroid dehydrogenases by treatment with a fish gonadotropin

Abstract In vitro steroidogenesis in the mature ovaries of a freshwater teleost, the ayu ( Plecoglossus altivelis ), was examined. Cell-free homogenates of the ovaries of untreated fish or fish treated with a salmon gonadotropin (SG-G100) were aerobically incubated with 14 C-labeled progesterone, 17α-hydroxyprogesterone, or 17α,20β-dihydroxy-4-pregnen-3-one, in the presence of NADPH. (1) Untreated fish: Progesterone was converted to 3α,17α-dihydroxy-5β-pregnan-20-one (the major product), 17α-hydroxyprogesterone, testosterone, 3α-hydroxy-5β-pregnan-20-one, 17α-hydroxy-5β-pregnane-3,20-dione, 17α,20β-dihydroxy-5β-pregnan-3-one, and 5β-pregnane-3α,17α,20β-triol. 17α-Hydroxyprogesterone was also transformed to 3α,17α-dihydroxy-5β-pregnan-20-one (the major product), in addition to the above-stated metabolites. 17α,20β-Dihydroxy-4-pregnen-3-one was metabolized into 17α,20β-dihydroxy-5β-pregnan-3-one and 5β-pregnane-3α,17α,20β-triol. (2) Fish treated with the gonadotropin: Besides the metabolites mentioned above, 17α,20β-dihydroxy-4-pregnen-3-one was confirmed as a metabolite of progesterone, and enhanced production of 17α,20β-dihydroxy-5β-pregnan-3-one and 5β-pregnane-3α,17α,20β-triol was observed. 17α-Hydroxyprogesterone was transformed to 17α,20β-dihydroxy-4-pregnen-3-one and 5β-pregnane-3β,17α,20β-triol, in addition to the metabolites obtained by the untreated fish, and formation of 17α,20β-dihydroxy-5β-pregnan-3-one and 5β-pregnane-3α,17α,20β-triol was increased. 17α,20β-Dihydroxy-4-pregnen-3-one was converted into 5β-pregnane-3β,17α,20β-triol besides 17α,20β-dihydroxy-5β-pregnan-3-one and 5β-pregnane-3α,17α,20β-triol. From these results it is concluded that, by the treatment of the fish with gonadotropin, ovarian 3β- and 20β-hydroxysteroid dehydrogenases were selectively activated.

In vitro bioconversions of steroids in the mature ovary of the hagfish, Eptatretus burgeri☆

Abstract 1. 1. Steroid hormone production in the mature ovary of the hagfish Eptatretus burgeri was demonstrated in vitro using labeled precursors of steroid hormones. 2. 2. Progesterone, 17α-hydroxyprogesterone, androstenedione and 11-deoxycortisol were identified as the metabolites, after incubation of the follicular tissue homogenate with 14 C-pregnenolone. Furthermore, 3α-hydroxy-5β-pregnan-20-one, as a metabolite of progesterone, and 5α-androstane-3,17-dione (5α-androstanedione) and 3β-hydroxy-5α-androstan-17-one (epiandrosterone), as metabolites of androstenedione, were identified respectively. 3. 3. From these identified metabolites, the metabolic pathways of steroids in the hagfish ovary are proposed. 4. 4. The relationship between the existence of 11-deoxycortisol and fish ovulation is discussed.