Yukinori Kazeto

Oceanic spawning ecology of freshwater eels in the western North Pacific

The natural reproductive ecology of freshwater eels remained a mystery even after some of their offshore spawning areas were discovered approximately 100 years ago. In this study, we investigate the spawning ecology of freshwater eels for the first time using collections of eggs, larvae and spawning-condition adults of two species in their shared spawning area in the Pacific. Ovaries of female Japanese eel and giant mottled eel adults were polycyclic, suggesting that freshwater eels can spawn more than once during a spawning season. The first collection of Japanese eel eggs near the West Mariana Ridge where adults and newly hatched larvae were also caught shows that spawning occurs during new moon periods throughout the spawning season. The depths where adults and newly hatched larvae were captured indicate that spawning occurs in shallower layers of 150–200 m and not at great depths. This type of spawning may reduce predation and facilitate reproductive success.

Characterization of a cDNA encoding P-450 aromatase (CYP19) from Japanese eel ovary and its expression in ovarian follicles during induced ovarian development.

A cDNA encoding P450 aromatase (CYP19) was isolated from a Japanese eel (Anguilla japonica) ovarian cDNA library. This cDNA contains a complete open reading frame encoding 511 amino acid residues. The deduced amino acid sequence is 59% and 65% identical to the catfish and rainbow trout forms, respectively, and 52-54% to mammalian and chicken forms. Non-steroidogenic COS-7 cells transfected with the eel CYP19 cDNA converted exogenous androstenedione to estrone, thus verifying its identity. Northern blot analysis indicated that there was a single 2.1 kb transcript in the ovary. A 2.1 kb transcript was also found in the brain but not in the spleen, head kidney, kidney, or liver. Throughout ovarian development induced by weekly injections of salmon pituitary homogenate (SPH, 20 microg/g body weight), the 2.1 kb transcript was barely or not detectable in the ovaries. However, signals greatly increased in intensity in oocytes in the migratory nucleus stage and then decreased slightly in the post-ovulatory ovary. These changes in transcript levels are consistent with the changes in aromatase activity of ovarian follicles, suggesting that aromatase activity in ovarian follicles is mainly regulated at the transcriptional level. In addition, fadrozole was found to significantly inhibit aromatase activity in a heterologous expression system using COS-7 cells, which indicates that fadrozole treatment could be useful to control E(2) production during artificial maturation of eels.

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

Abstract Serum levels of oestradiol-17β (E2), 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 E2 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 E2 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 E2, 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 E2, T and 17α,20β-DHP, and exogenous T to E2, 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 E2, T and 17α,20β-DHP in the absence of exogenous substrates was low. The ability of follicles to produce E2 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 E2 and aromatase activity (the ability of follicles to convert T to E2). E2 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.

Japanese Eel Follicle-Stimulating Hormone (Fsh) and Luteinizing Hormone (Lh): Production of Biologically Active Recombinant Fsh and Lh by Drosophila S2 Cells and Their Differential Actions on the Reproductive Biology

Abstract Two gonadotropins (Gths), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), control gonadal steroidogenesis and gametogenesis in vertebrates, including teleost fish. Here, we report on the production of biologically active recombinant Fsh (rec-Fsh) and Lh (rec-Lh) in Japanese eel using Drosophila S2 cells. The three subunits composing Gths, i.e., glycoprotein hormone, alpha polypeptide (Cga), follicle-stimulating hormone, beta polypeptide (Fshb), and luteinizing hormone, beta polypeptide (Lhb), were at first independently produced and were proven to be glycosylated and secreted as the mature peptides. Each beta subunit, along with its Cga, was simultaneously coexpressed to produce heterodimeric rec-Fsh and rec-Lh that were subsequently highly purified. The biological activity of rec-Gths was demonstrated in various in vitro assays. The rec-Gths differentially activated their receptors, which resulted in an increase in 11-ketotestosterone (11KT) secretion, a differential alteration of gene expression of steroidogenic enzymes in immature testis, and the induction of the complete process of spermatogenesis in vitro. The data strongly suggest that Fsh and Lh differentially play important roles in the reproductive physiology of the Japanese eel. By contrast, these rec-Gths exhibited little activity in the gonad when administered in vivo. This difference between in vitro and in vivo bioactivity is probably due to the qualitative nature of glycosylation in S2 cells, which resulted in degradation of the recombinant protein in vivo. These differences in the carbohydrate moieties need to be elucidated and ameliorated.

Oogenesis in the Japanese Eel, Anguilla japonica

Oogenesis in the teleost is generally divided into oocyte growth and maturation phases. The oocyte growth phase is further divided into primary and secondary growth phases, and the secondary growth phase consists of previtellogenic and vitellogenic phases. Both vitellogenic oocyte growth (vitellogenesis) and oocyte maturation, and possibly other phases as well, are under the control of pituitary gonadotropic hormone (GTH). However, in most cases, GTH action is not direct, but exerts its influence through the production of sex steroid hormones. During the period of vitellogenesis, ovarian follicle cells produce estrogen, mainly 1713estradiol (E2), to stimulate hepatic synthesis of a yolk precursor protein, vitellogenin (VTG). VTG is incorporated into the oocyte and reconstructed as a yolk protein (Wallace 1985). After the completion of oocyte growth, ovarian follicle cells produce maturation-inducing hormone (MIH) to induce oocyte maturation directly (Nagahama 1987). Oocyte maturation is one of the most important steps in acquiring the competency to undergo fertilization and consists of the breakdown of the germinal vesicle (GVBD), chromosome condensation, and extrusion of the first polar body. Oocyte meiosis is reinitiated by stimulation with MIH and is then arrested at metaphase in the second meiotic division. Subsequently, the oocyte is ovulated as a fertilizable egg. In teleosts, either 17α,20β-dihydroxy-4-pregnen-3one (DHP) or 17α,20β, 21-trihydroxy-4-pregnen-3-one (20β-S) have been known as MIH (Nagahama and Adachi 1985; Trant and Thomas 1989).

Synthesis and possible function of 11-ketotestosterone during oogenesis in eel (Anguilla spp.)

The control of 11-ketotestosterone (11-KT) biosynthesis and its physiological roles were examined in female Japanese eel (Anguilla japonica) and New Zealand longfinned eel (Anguilla dieffenbachii). 11-KT was detected in serum of female eels of both species. Among various tissues from Japanese eel, the ovary had the greatest capacity to synthesize 11-KT in vitro. In addition, the oocyte diameters of eels treated with 11-KT had increased significantly. Furthermore, these oocytes were found to have an increased number of oil droplets. These findings suggest that 11-KT in female eels may be mostly of ovarian origin and that this androgen appears to play an important role in controlling pre-vitellogenic oocyte growth.

Cloning and characterization of a cDNA encoding cholesterol side-chain cleavage cytochrome P450 (CYP11A1): Tissue-distribution and changes in the transcript abundance in ovarian tissue of Japanese eel, Anguilla japonica, during artificially induced sexual development

Cholesterol side-chain cleavage cytochrome P450 (CYP11A1: P450scc) is a crucial steroidogenic enzyme that catalyzes an initial step in the production of all classes of steroids. A cDNA encoding Japanese eel P450scc was cloned and characterized. The cDNA putatively encoded 521 amino acid residues with high homology to those of other vertebrate forms. The recombinant P450scc produced in COS-7 cells efficiently catalyzed the conversion of 25-hydroxycholesterol into pregnenolone. By northern blot, a single P450scc transcript of approximately 3.3 kb was detected in both ovary and head kidney. Transcript levels of this enzyme significantly increased throughout ovarian development artificially induced by salmon pituitary homogenate, which suggests that gonadotropic stimuli can induce ovarian expression of the P450scc gene in teleosts, as has been reported in mammals. Furthermore, RT-PCR analysis revealed that gene expression of three steroidogenic enzymes, P450scc, P450c17 and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) show distinctly different tissue-specific patterns of expression in the Japanese eel. The P450scc gene was expressed in ovary and head kidney while the sole source of the P450c17 transcript was ovary. In contrast, 3beta-HSD transcript was detected in all tissues examined, brain, liver, spleen and trunk kidney, etc. These suggest that some steroidogenic enzymes are also expressed in non-endocrine tissues and could potentially regulate the local and/or circulating steroid levels in teleosts, as they do in mammals.

Ovarian steroidogenesis and the role of sex steroid hormones on ovarian growth and maturation of the Japanese eel

Three sex steroid hormones, estradiol-17β (E2), 11-ketotestosterone (11-KT), and 17α,20β-dihydroxy-4-pregnen-3-one (DHP), are well established as primary estrogen, androgen, and progestin, respectively, in teleost fish. Japanese eel, Anguilla japonica, would be a suitable candidate to study ovarian steroid physiology of fish because the ovarian growth and steroidogenesis is dormant under laboratory condition but can be induced by administration of exogenous gonadotropic reagents. In this review, we summarized our work on the function and production of sex steroid hormones in the ovary of the Japanese eel during ovarian growth and oocyte maturation artificially induced by treatment with extract of salmon pituitary. In vitro and in vivo assays suggest that 11-KT and E2 play primary roles in previtellogenic and vitellogenic growth of oocytes, respectively, whereas DHP is essential for induction of final oocyte maturation. We also reviewed the correlation between ovarian steroidogenesis to produce these sex steroid hormones, serum titers and gene expression.

Expression of androgen receptor mRNA in the ovary of Japanese eel, Anguilla japonica, during artificially induced ovarian development

In order to elucidate how androgens may mediate their effects on ovarian growth, we investigated the mRNA levels of two subtypes of androgen receptor (ara and arb) in the ovary of feminized Japanese eel (Anguilla japonica) during artificially induced ovarian development by quantitative real-time reverse transcriptase polymerase chain reaction and in situ hybridization. Ara mRNA levels were high from the late oil droplet stage to the late vitellogenic stage, whereas arb mRNA levels were high from the late oil droplet stage to the midvitellogenic stage. Both ar mRNAs were predominantly observed in the follicle cells and the epithelial cells of the ovigerous lamellae in all stages. In the oil droplet stage, oogonia exhibited intense signals for ar mRNAs. There was no obvious difference in localization pattern between ara and arb in all ovaries examined, irrespective of maturational stage. It was difficult to identify the follicle cell types that were positive for ar mRNA during ovarian development. Only in post-ovulatory follicles could theca and granulosa cells be clearly identified, and ar signals were observed in both layers. The predominant localization of ar mRNA in the follicle cells suggests that androgens play important roles in oocyte growth by acting on these cells in this species. We have shown the expression profile and localization of ar mRNA during ovarian development for the first time in an oviparous vertebrate.

Androgen-specific regulation of FSH signalling in the previtellogenic ovary and pituitary of the New Zealand shortfinned eel, Anguilla australis.

The evidence for androgens having a pivotal role in the functioning of the female reproductive axis--such as initiating puberty or vitellogenesis--is mounting. However, the use of aromatizable androgens and the tissue-specific focus of most studies often make it unclear if androgenic effects throughout the axis proceed via androgen or estrogen signalling mechanisms. In this study, we assessed the effects of 11-ketotestosterone (11KT, a non-aromatizable androgen) on the pituitary and ovary of previtellogenic (PV) freshwater eels Anguilla australis, comparing them with eels naturally undergoing early vitellogenesis (EV). We found that 11KT treatment produces molecular and morpho-physiological phenotypes that were generally intermediate between PV and EV. Most notably, we demonstrated that 11KT induces effects on follicle-stimulating hormone (FSH) signalling in the pituitary and ovaries that are in opposition to each other. Thus, 11KT significantly reduced fshβ subunit expression in the pituitary. At the same time, 11KT dramatically increased mRNA levels of ovarian FSH receptor and plasma levels of estradiol-17β, very likely sensitizing the previtellogenic follicle to the FSH signal. Androgens therefore may be important in facilitating puberty in the eel.