Shigeho Ijiri

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.

Changes in the Expression of Genes Encoding Steroidogenic Enzymes in the Channel Catfish (Ictalurus punctatus) Ovary Throughout a Reproductive Cycle

Abstract In vertebrates, the growth and maturation of the ovarian follicle is dependent on the appropriate dynamics of sex steroid secretion, which is dictated by gene expression of the steroidogenic enzymes. The molecular aspects of steroid regulation are poorly understood in fishes, so as a first step we determined the pattern of expression of four key steroidogenic genes throughout the ovarian cycle in an annually spawning teleost, the channel catfish (Ictalurus punctatus). The abundance of transcripts encoding 3β-hydroxysteroid dehydrogenase (3β-HSD) and cholesterol side chain cleavage (P450scc), 17α-hydroxylase/lyase (P450c17), and aromatase (P450arom) were determined by rtqRT-PCR or ribonuclease protection assay and correlated to ovarian growth and plasma titers of estradiol (E2) and testosterone (T) in two populations of catfish. Elevations in transcript abundance for P450c17, P450scc, and P450arom were observed at the onset of ovarian recrudescence and during early vitellogenic growth of the oocytes; however, all three decreased precipitously with the completion of vitellogenesis. Changes in the expression of these genes strongly suggest a direct correlation to E2 and T titers. Alternatively 3β-HSD transcript abundance was relatively stable throughout the year. This study suggests that the genes encoding the three steroidogenic cytochrome P450s have a similar regulatory mechanism.

Molecular Biology of the Channel Catfish Gonadotropin Receptors: 2. Complementary DNA Cloning, Functional Expression, and Seasonal Gene Expression of the Follicle-Stimulating Hormone Receptor

Abstract Molecular cloning of the channel catfish FSH receptor is reported together with temporal changes in the gene expression throughout a reproductive cycle. A cDNA encoding the receptor was isolated from the testis using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) procedures. The cDNA coded for a 662-amino acid protein that was most identical (51%–59%) to salmon gonadotropin receptor I and the FSH receptors of higher vertebrates, and less identical to LH receptors and thyrotropin receptors (45%–49% and 46%–47%, respectively). In addition, PCR analysis of the genomic DNA showed the absence of the LH receptor-specific intron. Expression of the channel catfish FSH receptor gene was highly restricted to the testis and ovary, except for a low-level expression in the spleen. Transfected COS cells expressed an active recombinant receptor as determined by the ligand-specific activation of a cAMP-responsive reporter gene (luciferase). The recombinant receptor was activated by human FSH and, to a small extent, hCG. Seasonal changes in the ovarian expression of the FSH receptor gene, examined by measuring the transcript abundance by quantitative real-time RT-PCR, showed a rise around the time of onset of ovarian recrudescence and a decrease prior to spawning. This pattern of seasonal expression of FSH receptor differs significantly from that of the LH receptor, which we reported recently. The differential expression of the two gonadotropin receptor genes, in addition to the differential secretion of the gonadotropic hormones, seem to be critical for the regulation of steroidogenesis and other gonadal physiological processes.

Molecular characterization of the GnRH system in zebrafish (Danio rerio): cloning of chicken GnRH-II, adult brain expression patterns and pituitary content of salmon GnRH and chicken GnRH-II.

The zebrafish has proven to be a model system with unparalleled utility in vertebrate genetic and developmental studies. Substantially less attention has been paid to the potential role that zebrafish can play in answering important questions of vertebrate reproductive endocrinology. As an initial step towards exploiting the advantages that the zebrafish model offers, we have characterized their gonadotropin-releasing hormone (GnRH) system at the molecular level. GnRHs comprise a family of highly conserved decapeptide neurohormones widely recognized to orchestrate the hormonal control of reproduction in all vertebrates. We have isolated the gene and cDNA encoding chicken GnRH-II (cGnRH-II) from zebrafish, as well as several kilobases of upstream promoter sequence for this gene. As the gene encoding salmon GnRH (sGnRH) has been previously isolated (Torgersen et al, 2002), this is the second GnRH gene isolated from zebrafish to date. We have localized expression of these two genes in the brains of reproductively mature zebrafish using in situ hybridization. sGnRH is localized to the olfactory bulb-terminal nerve region (OB-TN), the ventral telencephalon-preoptic area (VT-POA) and, as we report here for the first time in any teleost species, the hindbrain. cGnRH-II is expressed exclusively in the midbrain, as has been found in all other jawed vertebrate species examined. Finally, the levels of both GnRH peptides in pituitaries of reproductively mature zebrafish were quantified using specific ELISAs. sGnRH pituitary peptide levels were shown to be 3- to 4-fold higher than cGnRH-II pituitary peptide. The cumulative results of these experiments allow us to conclude that zebrafish express just two forms of GnRH in a site-specific manner within the brain, and that sGnRH is the hypophysiotropic GnRH form. This work lays the foundation for further research into the control of reproduction in zebrafish, such as the functional significance of multiple GnRHs in vertebrates, and the molecular mechanisms controlling tissue-specific GnRH expression.

Molecular Biology of Channel Catfish Gonadotropin Receptors: 1. Cloning of a Functional Luteinizing Hormone Receptor and Preovulatory Induction of Gene Expression

Abstract There is little known about the molecular biology of piscine gonadotropin receptors, and information about gene expression during reproductive development is particularly lacking. We have cloned the LH receptor (LHR) in the channel catfish (cc), and examined its gene expression throughout a reproductive cycle. A cDNA encoding the receptor was isolated from the testis using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends procedures. It encoded a 696-amino acid protein that showed the greatest homology (46–50% identity) with the known LHRs and lesser similarity with FSH receptors and thyroid-stimulating hormone receptors (44–47% and 42–44% identity, respectively). In addition, two characteristics unique to the LHRs were conserved in the cloned receptor and the encoding gene: presence of an intron corresponding to intron 10 in mammals and turkey and occurrence of a double cysteine residue in the cytoplasmic tail for potential palmitoylation. The ccLHR gene was well expressed in the gonads and kidney and merely detectable in the gills, muscle, and spleen. The isolated cDNA encoded an active ccLHR protein, as the recombinant receptor expressed in COS7 cells activated a cAMP response element-driven reporter gene (luciferase) upon exposure to hCG in a dose-dependent manner. Seasonal changes in the ovarian expression of the ccLHR gene, as examined by measuring the transcript abundance by quantitative real-time RT-PCR, remained rather low during most of the reproductive cycle but was acutely induced around the time of spawning. This pattern of expression correlates well with the reported expression of its ligand (LH) in fishes and concurs with the notion that LH is a key regulator of the periovulatory maturational events.

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.

Cloning and functional expression of a thyrotropin receptor from the gonads of a vertebrate (bony fish): potential thyroid-independent role for thyrotropin in reproduction.

The thyroid stimulating hormone receptor (TSHR) mediates the pituitary control of the development, growth and function of the thyroid. The expression of the gene encoding this receptor is known only in the thyroid, lymphocytes, fibroblasts, retro-orbital tissue and fat cells. We have cloned a TSHR from the gonads of a non-mammalian vertebrate, a bony fish (striped bass, Morone saxatilis) in the course of our search for gonadotropin receptors (follicle stimulating hormone receptor, FSHR and luteinizing hormone receptor, LHR). RT-PCR analysis demonstrated that the striped bass TSHR (stbTSHR) transcripts were abundant in both the thyroid and gonads and detectable in skeletal muscle, heart and brain tissues. The stbTSHR cDNA encoded a 779-amino acid glycoprotein hormone receptor with much higher homology (57-59%) to the mammalian TSH receptors than the mammalian LH receptors (47-49%) and FSH receptors (47%), and salmon and catfish gonadotropin receptors (42-45%). There was a TSHR-specific insertion in the extracellular domain as seen in mammalian receptors. Moreover, PCR analysis of genomic DNA indicated the absence of the LHR-specific intron in the striped bass TSHR gene. Recombinant stbTSHR expressed in COS1 cells activated reporter genes (luciferase) driven by either a cAMP response element or the c-fos promoter in response to bovine TSH, stbLH or hCG, but not human FSH. In situ hybridization studies revealed the presence of stbTSHR transcripts in the gametes but not in the follicular cells. This pattern of expression is unique and suggests a direct, albeit unknown, role for TSH in gamete physiology.

Molecular Biology of Ovarian Aromatase in Sex Reversal: Complementary DNA and 5′-Flanking Region Isolation and Differential Expression of Ovarian Aromatase in the Gilthead Seabream (Sparus aurata)

Abstract To elucidate the involvement of aromatase in sex reversal, the gilthead seabream ovarian P450 aromatase (cyp19a1a) cDNA and its 5′-flanking region were isolated and characterized. Northern blot analysis revealed that only one cyp19a1a transcript (2.0 kb) is expressed in the ovary. Four cAMP-responsive elements were identified at the 5′-flanking region of seabream cyp19a1a indicating a high potential to respond to gonadotropin signaling. Studying the seasonal profile, two expression peaks of cyp19a1a transcripts in the ovarian tissues were found in July (about 15000 copies/ng total RNA) for ambisexual fish and in December (about 12000 copies/ng total RNA) for spawning females. Starting from September, transcript levels of cyp19a1a in the ovarian portions of the male-developing gonads gradually decreased. Furthermore, the ovarian portions of the female gonads expressed cyp19a1a at a significantly higher level than the ovarian portions of the male gonads after November. Taken together with levels of plasma estradiol in reversing females being significantly higher than those in developing males, the above results reinforce the importance of cyp19a1a in sex reversal. In vitro exposure of ovarian fragments to gonadotropins (hCG) at 1, 10, and 100 IU/ml significantly (P < 0.05) upregulated cyp19a1a expression. Additionally, expression of cyp19a1a displayed a stronger and significant correlation with the transcript expression of ovarian Lh receptor rather than Fsh receptor during the ambisexual stage. Our results indicate that the differential expression of cyp19a1a gene is associated with sex reversal and that gonadotropin signals (particularly Lh) may serve as major players in regulating the expression of cyp19a1a during the process of sex reversal.

Androgens and Very Low Density Lipoprotein Are Essential for the Growth of Previtellogenic Oocytes from Japanese Eel, Anguilla japonica, In Vitro

To investigate the regulation of lipid uptake into the eel oocyte in more detail, effects of 11-ketotestosterone (11-KT) and lipid transporters (lipoproteins) were determined in vitro. Ovarian explants from previtellogenic Japanese eels (Anguilla japonica) were incubated for 28 days with 11-KT and/or with very low density lipoproteins (Vldl), low density lipoproteins (Ldl), or high density lipoproteins (Hdl) purified from eel plasma. The androgen 11-KT induced notable increases in oocyte diameter, which were accompanied by the appearance of vacuoles rather than lipid. Ldl and Hdl increased oocyte diameters, whereas Vldl did not. However, coincubation of 11-KT and Vldl, but not of Ldl or Hdl, resulted in dramatic increases in oocyte size and lipid droplet surface area. Effects of both 11-KT (oocyte size) and Vldl (lipid droplet surface area) were dose-dependent between 1 and 100 ng/ml and between 0.5 and 5 mg/ml, respectively. Interestingly, abnormal oocyte cytology under conditions of coculture with 11-KT and Vldl could essentially be prevented if Vldl concentrations were high enough (≥ 5 mg Vldl/ml medium). Unlike 11-KT, estradiol-17beta had no effect on oocyte diameter or lipid droplet surface area. We conclude that Vldl is a key transporter of neutral lipids that accumulate into the eel oocyte during oogenesis and that Vldl-dependent lipid uptake is stimulated by the androgen 11-KT.