Shinji Adachi

Identification of maturation-inducing steroid in a teleost, the amago salmon (Oncorhynchus rhodurus)

Maturation-inducing steroid in amago salmon (Oncorhynchus rhodurus) has been identified from media in which immature but fully grown folliculated oocytes of amago salmon had been incubated for 18-24 hr with chum salmon gonadotropin (SGA). The maturation-inducing (MI) activity of residues at various steps of purification was assessed by an in vitro germinal vesicle breakdown (GVBD) assay based on fully grown prophase-arrested folliculated oocytes of amago salmon. Ether extracts of the media from these incubates showed high MI activity. Yolk and oil droplets were removed from the ether extract by partition with equal volumes of 50% methanol and n-hexane. MI activity was found only in the 50% methanol phase. The 50% methanol phase was then fractionated (20 separate fractions) by reversed-phase high-performance liquid chromatography. MI activity was found only in fraction 10 which had a retention time coinciding exactly with 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHprog). The purity and final characterization of the residues of fraction 10 were further confirmed by thin-layer chromatography and mass spectrometry with authentic 17 alpha,20 beta-diOHprog standard. The present study, together with our previous findings that in amago salmon 17 alpha,20 beta-diOHprog is the most potent steroid for the induction of oocyte maturation in vitro and is present at high concentrations in the plasma only around the time of oocyte maturation, indicates that 17 alpha,20 beta-diOHprog is the major naturally occurring maturation-inducing steroid in this species.

Estradiol-17β production in amago salmon (Oncorhynchus rhodurus) ovarian follicles: Role of the thecal and granulosa cells

Separation of the follicular thecal cell and granulosa cell layers of oocytes of the amago salmon (Oncorhynchus rhodurus) facilitated assessment of their roles in estradiol-17β production by an in vitro incubation method. Four different follicular preparations, intact follicles (oocytes with complete follicle layers), thecal layers contaminated with less than 10% granulosa cells, pure granulosa layers and zona radiata, and thecal layer-granulosa layer co-cultures, were incubated in the presence or absence of partially purified chinook salmon gonadotropin (SG-G100). Estradiol-17β and testosterone levels in the medium were measured by specific radioimmunoassay. SG-G100 (0.1, 1 μg/ml) stimulated estradiol-17β production by intact follicles and co-culture preparations, but not by the isolated thecal or granulosa layers, indicating that both layers are necessary for gonadotropin-stimulated estradiol-17β production. In contrast, SG-G100 greatly stimulated testosterone production by thecal layers (up to 80 times basal control values) but only slightly stimulated testosterone production by the other follicular preparations. Incubation of granulosa layers with exogenous testosterone (0.01, 1 μg/ml) or with testosterone and SG-G100 (1 μg/ml) together resulted in elevated testosterone levels. No significant difference was found between the two treatments, suggesting that gonadotropin did not stimulate the conversion of testosterone to estradiol-17β. Isolated thecal layers incubated with testosterone produced relatively small amounts of estradiol-17β, 7–8 times less than granulosa layers under the same conditions. These results suggest a two-cell-type model for the production of follicular estrogens, the thecal layer possibly contributing to estradiol-17β production by synthesizing androgens which are transferred to the granulosa layer and aromatized to estradiol-17β.

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.

Diurnal rhythm of oocyte development and plasma steroid hormone levels in the female red sea bream, Pagrus major, during the spawning season

Abstract Diurnal maturation rhythm in the female red sea bream, Pagrus major , which spawns every day during the spawning season and has an asynchronous-type ovary containing oocytes at various stages of development, was studied by histological investigation of the ovary. Plasma levels of estradiol-17β and testosterone corresponding to the ovarian maturation rhythm were also investigated. The fish had a diurnal ovarian maturation rhythm in which the most advanced oocytes in the ovary finished germinal vesicle breakdown at 07.00 h, arrived at a mature state at 10.00 h, and ovulation began at 13.00 h. Spawning mainly occurred between 18.00 and 19.00 h. Plasma estradiol-17β levels reached a peak of about 1200 pg/ml in fish with pre-mature stage oocytes at 07.00 h, followed by a sharp drop in ovulated fish at 13.00 h (to about 200 pg/ml); the levels showed a tendency to increase from the time of postovulation to the next germinal vesicle breakdown. Testosterone levels decreased (to about 20 pg/ml) at ovulation time (13.00 h) in parallel with the estradiol-17β levels, but were largely unchanged (70–110 pg/ml) throughout the rest of the ovarian maturation cycle. These results are discussed in relation to other studies on the role of these steroid hormones responsible for the ovarian maturation of teleosts.

Rainbow trout cytochrome P‐450c17 (17α‐hydroxylase/17,20‐lyase) cDNA cloning, enzymatic properties and temporal pattern of ovarian P‐450c17 mRNA expression during oogenesis

A cDNA clone encoding cytochrome P‐450c17 (17α‐hydroxylase/17,20‐lyase) was isolated from a rainbow trout ovarian follicle cDNA library. The cDNA contained an open reading frame of 1,542 nucleotides encoding a protein of 514 amino acid residues. The amino acid sequence of trout P‐450c17 shows a much greater homology with chicken P‐450c17 than with that of human, bovine and rat. The trout P‐450c17 expressed in non‐steroidogenic mammalian COS‐1 cells showed both 17α‐hydroxylase and 17,20‐lyase activities. The cDNA only hybridized to a single species or mRNA (2.4 kb) isolated from rainbow trout ovaries; the 2.4 kb transcripts were abundant in trout ovaries during the later stages of oogenesis.

Role of ovarian thecal and granulosa layers in gonadotropin-induced synthesis of a salmonid maturation-inducing substance (17α,20β-dihydroxy-4-pregnen-3-one)☆

The role of the thecal and granulosa layers of the ovarian follicle of the rainbow trout (Salmo gairdneri) in the production of maturation-inducing substance, 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-diOHprog) was studied using isolated follicular components in vitro. Isolated thecal layers, intact follicles, and cocultures of isolated thecal and granulosa layers produced substantial amounts of 17α-hydroxyprogesterone in response to stimulation with a partially purified chinook salmon gonadotropin (SG-G100) for 12 hr. However, after 24 and 48 hr incubation significantly less 17α-hydroxprogesterone was present in media from incubations of intact follicles and coculture preparations. SG-G100 did not stimulate 17α-hydroxyprogesterone production by granulosa layers. SG-G100 stimulated production of 17α,20β-diOHprog by thecal layers, intact follicles and coculture preparations but not by granulosa layers. Levels in media from the thecal layer incubations remained low and constant throughout the 48-hr incubation period, but levels in media from incubations of intact follicles and coculture preparations peaked at 24 hr, concomitant with the sharp decline in 17α-hydroxyprogesterone levels, suggesting that thecal layers produce 17α-hydroxyprogesterone which is converted to 17α,20β-diOHprog by granulosa cells. 17α,20β-DiOHprog was produced by granulosa layers when they were incubated with 17α-hydroxyprogesterone; SG-G100 significantly increased the production of 17α,20β-diOHprog, indicating an enhancement of the activity of the enzyme 20β-hydroxysteroid dehydrogenase (20β-HSD) by the gonadotropin. Both dibutyryl cAMP and forskolin, an adenylate cyclase activator, had effects similar to partially purified chum salmon gonadotropin (SGA) suggesting that the gonadotropin-induced enhancement of 20β-HSD involves a cAMP-dependent step. Based on these data a two-cell model for maturation-inducing substance production is proposed, in which gonadotropin acts on both the thecal and granulosa layers to stimulate 17α-hydroxyprogesterone production and 20β-HSD activity, respectively.

Developmental changes in steroidogenic responses of ovarian follicles of amago salmon (Oncorhynchus rhodurus) to chum Salmon gonadotropin during oogenesis

Changes in amago salmon (Oncorhynchus rhodurus) ovarian thecal and granulosa layer function in association with the production of two biologically important ovarian mediators of oocyte growth and maturation in salmonids, estradiol-17 beta and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHprog), were investigated using isolated follicular preparations in vitro. A distinct shift of steroidogenic responses of intact follicles from estradiol-17 beta to 17 alpha,20 beta-diOHprog in response to partially purified chum salmon gonadotropin (SGA) occurred immediately prior to oocyte maturation. Aromatase activity in granulosa layers increased during vitellogenesis and decreased rapidly prior to oocyte maturation. This decrease in aromatase activity was coincident with the decreased ability of intact follicles to produce estradiol-17 beta in response to SGA. Since testosterone production in thecal layers did not decline during this time, the reduced production of estradiol-17 beta by postvitellogenic follicles is due, in part, to decreased aromatase activity in granulosa layers. Immediately prior to oocyte maturation, intact follicles acquire an increased ability to produce 17 alpha,20 beta-diOHprog in response to SGA. Although granulosa layers first acquired the ability to convert exogenous 17 alpha-hydroxyprogesterone (17 alpha-OHprog) to 17 alpha,20 beta-diOHprog (20 beta-hydroxysteroid dehydrogenase, 20 beta-HSD, activity) in response to SGA about 2 months prior to oocyte maturation, thecal layers did not develop the ability to produce 17 alpha-OHprog in response to SGA until immediately prior to oocyte maturation. Thus, changes in thecal cell function are critical for intact follicles to acquire the ability to produce 17 alpha,20 beta-diOHprog in response to gonadotropin.

Some endocrine factors affecting the development of seawater tolerance during the parr-smolt transformation of the amago salmon, Oncorhynchus rhodurus

Abstract The development of hypoosmoregulatory abilities assessed by monthly seawater-challenge tests was studied in hatchery-reared amago salmon. At any time from August to May, parr, including precociously mature male and immature small fish, could not regulate serum sodium levels 24 h after transfer to seawater (29‰). The best hypoosmoregulatory performance was obtained from smolts collected in December and January, and this coincided with the peak of smoltification assessed by external appearance. The hypoosmoregulatory capacity of smolts began to decline in February and reached the same levels as parr by May, thus indicating that desmoltification was occurring during the spring. Analysis of weekly plasma samples between 30 August and 14 November demonstrated that the levels of plasma thyroxine of smolts increased significantly just prior to the maximum development of seawater adaptability; parr did not show this increase. The greatest thyroid activity, assessed by changes in thyroid epithelial cell height, was observed in smolts collected in December and January. These results suggest that thyroid hormone is involved in smoltification in the amago salmon. Possible relationships between plasma thyroxine and sex steroid levels in precociously mature male parr and smolts are discussed.

Rainbow trout ovarian cholesterol side-chain cleavage cytochrome P450 (P450scc): cDNA cloning and mRNA expression during oogenesis

A cDNA clone encoding cholesterol side‐chain cleavage cytochrome P450 (P450scc) was isolated from a rainbow trout ovarian follicle cDNA library. The cDNA contains an open reading frame of 1,542 nucleotides encoding a protein of 514 amino acids. The predicted amino acid sequence of trout P450scc shows 48% homology with that of human, and 46% homology with that of rat, bovine and pig. P450scc activity was confirmed by transfected COS‐1 monkey kidney tumour cells with an expression vector for trout P450scc cDNA and subsequent detection of conversion from 25‐hydroxycholesterol to pregnenolone by radioimmunoassay. The cDNA only hybridized to a single 1.8 kb RNA transcript. The transcript was not found in early vitellogenic follicles, barely detected in postvitellogenic follicles, and abundant in postovulatory follicles.

Diurnal rhythm of serum steroid hormone levels in the Japanese whiting, Sillago japonica, a daily-spawning teleost

Ovarian developmental stages and serum steroid hormone levels were examined at six different times of day (0100, 0600, 1000, 1300, 1600, 2000 h) in a marine teleost, the Japanese whiting Sillago japonica, which has an asynchronous-type ovary containing oocytes at various stages of development and spawns every day during a period ranging up to three months. The largest oocytes in the ovaries at the active vitellogenic or post-vitellogenic stages were found between 0100 and 1300 h. Oocyte maturation indicated by germinal vesicle breakdown (GVBD) occurred at 1600 h, and ovulated oocytes were observed in the ovaries collected at 2000 h. These processes were accompanied by a significant daily change in serum steroid hormone levels. The serum level of estradiol-17β showed a peak in fish with mature oocytes sampled at 1600 h. In these fish, the second-largest oocytes in the ovaries were at the initial stage of vigorous vitellogenesis, the secondary yolk stage. Therefore the highest level of serum estradiol-17β was considered to be due to the second-largest oocytes. Testosterone levels remained low and constant throughout the experimental period. The serum levels of 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-diOHprog) peaked at 1600 h at which time all fish had mature oocytes. These results indicate that the Japanese whiting possesses a diurnal rhythm of oocyte development including vitellogenesis, oocyte maturation and ovulation, and further suggest that daily cycles in oocyte growth and maturation which simultaneously take place in an ovary are regulated by diurnal secretions of estradiol-17β and the maturation-inducing steroid, 17α,20β-diOHprog.