Language：Japanese   Publishing type：Research paper (conference, symposium, etc.)  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Background: Spermatogonia are highly tolerant to reactive oxygen species (ROS) attack while advanced-stage germ cells such as spermatozoa are much more susceptible, but the precise reason for this variation in ROS tolerance remains unknown.
Methodology/Principal Findings: Using the Japanese eel testicular culture system that enables a complete spermatogenesis in vitro, we report that advanced-stage germ cells undergo intense apoptosis and exhibit strong signal for 8-hydroxy-29-deoxyguanosine, an oxidative DNA damage marker, upon exposure to hypoxanthine-generated ROS while spermatogonia remain unaltered. Activity assay of antioxidant enzyme, superoxide dismutase (SOD) and Western blot analysis using an anti-Copper/Zinc (Cu/Zn) SOD antibody showed a high SOD activity and Cu/Zn SOD protein concentration during early spermatogenesis. Immunohistochemistry showed a strong expression for Cu/Zn SOD in spermatogonia but weak expression in advanced-stage germ cells. Zn deficiency reduced activity of the recombinant eel Cu/Zn SOD protein. Cu/Zn SOD siRNA decreased Cu/Zn SOD expression in spermatogonia and led to increased oxidative damage.
Conclusions/Significance: These data indicate that the presence of high levels of Cu/Zn SOD and Zn render spermatogonia resistant to ROS, and consequently protected from oxidative stress. These findings provide the biochemical basis for the high tolerance of spermatogonia to oxidative stress.

DOI： 10.1371/journal.pone.0016938

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOSCIENTIFICA LTD  

The precise mechanism and direct effects of arsenic on fish, particularly in reproduction, are not well clarified. The aim of this study is to investigate the direct influence of arsenic on fish spermatogenesis using the Japanese eel (Anguilla japonica) in vitro testicular organ culture system. Eel testicular fragments were cultured in vitro with 0.1-100 mu M arsenic with or without human chorionic gonadotropin (hCG) for 6 or 15 days at 20 degrees C. Arsenic treatment provoked a dose-dependent inhibition of hCG-induced germ cell proliferation as revealed by 5-bromo-2-deoxyuridine immunohistochemistry. Time-resolved fluorescent immunoassay showed that arsenic suppressed hCG-induced synthesis of 11-ketotestosterone (11-KT) in testicular fragments incubated with 0.0001-100 mu M arsenic and hCG for 18 h. A 0.1 mu M (7 mu g/l) dose of arsenic which is lower than the World Health Organization drinking water quality guideline of 10 mu g/l most effectively reduced 11-KT production. The hCG-induced synthesis of progesterone from pregnenolone was significantly inhibited by low doses of arsenic (0.1-1 mu M), implying an inhibition of 3 beta-hydroxysteroid dehydrogenase activity. In situ TUNEL assays indicated that germ cells undergo apoptosis at the highest dose of arsenic (100 mu M). An arsenic concentration-dependent increase in oxidative DNA damage was detected by 8-hydroxy-2'-deoxyguanosine (8-OHdG) immunohistochemistry. A peak in 8-OHdG index was observed in testicular fragments treated with 100 mu M arsenic and hCG consistent with the TUNEL results. These data suggest that low doses of arsenic may inhibit spermatogenesis via steroidogenesis suppression, while high doses of arsenic induce oxidative stress-mediated germ cell apoptosis. Reproduction (2009) 138 279-287

DOI： 10.1530/REP-09-0167

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Zinc (Zn) plays important roles in various biological activities but there is little available information regarding its functions in spermatogenesis. In our current study, we further examined the role of Zn during spermatogenesis in the Japanese eel ( Anguilla japonica). Human CG (hCG) was injected into the animals to induce spermatogenesis, after which the concentration of Zn in the testis increased in tandem with the progression of spermatogenesis. Staining of testicular cells with a Zn-specific fluorescent probe revealed that Zn accumulates in germ cells, particularly in the mitochondria of spermatogonia and spermatozoa. Using an in vitro testicular organ culture system for the Japanese eel, production of a Zn deficiency by chelation with N,N,N',N'-tetrakis (2-pyridylemethyl) ethylenediamine ( TPEN) caused apoptosis of the germ cells. However, this cell death was rescued by the addition of Zn to the cultures. Furthermore, an induced deficiency of Zn by TPEN chelation was found to inhibit the germ cell proliferation induced by 11-ketotestosterone (KT), a fish specific androgen, 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP), the initiator of meiosis in fish, and estradiol-17 beta (E2), an inducer of spermatogonial stem-cell renewal. We also investigated the effects of Zn deficiency on sperm motility and observed that TPEN treatment of eel sperm suppressed the rate and duration of their motility but that co-treatment with Zn blocked the effects of TPEN. Our present results thus suggest that Zn is an essential trace element for the maintenance of germ cells, the progression spermatogenesis, and the regulation of sperm motility.

DOI： 10.1073/pnas.0900602106

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Importin alpha proteins are critical modulators of the classical nuclear protein import pathway. Although the physiological roles of importin alpha have been extensively studied in invertebrates and mammals, very little is known about their counterparts in lower vertebrates. In this study, to elucidate the roles of importin a in a teleost species, we isolated and characterized red seabream (Pagrus major) importin a cDNA derived from ovary and found changes in the mRNA levels of importin alpha in male and female red seabream during sexual maturation. The 1846-bp cDNA encodes a 520 amino acid protein that includes the importin 3-binding domain, a short acidic domain, and an armadillo (arm) repeat domain. Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) showed transcription of red seabream importin a in testis and ovary but not in the other tissues. The importin a mRNA levels in males increase in association with testicular development, whereas those in females remain high throughout sexual maturation. These findings suggest that red seabream ovary-derived importin a may be controlled in a tissue-specific manner and may perform unique functions in the gonad in addition to its involvement in nuclear transport. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cbpb.2008.08.010

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

To estimate the influence of water contamination by arsenic (As) on reproduction of crustaceans in Vietnam, we collected wild freshwater crab Somanniathelphusa pax from the Mekong Delta area in Vietnam, investigated gonadal development, and measured As concentration in hepatopancreas. In female crab, vitellogenesis was delayed in association with the increase of As accumulation in hepatopancreas, whereas there was no significant correlation between testicular development and As accumulation in male crab. To clarify the effects of As on gonadal development of crustaceans, we investigated the effects of oral As administration on gonadal development in Japanese freshwater crab Geothelphusa dehaani. In male crab, the occurrence of spermatids and spermatozoa were predominantly observed in the control group, whereas the occurrence of spermatocytes increased after administration of 10 mu g/crab As for 3 months. On the other hand, in females, secondary yolk globule stages mainly occupied ovary of the control group. However, the primary yolk globule stage gradually increased after 10 mu g/crab As administration. Together these results indicate that it is possible that As contamination in water or food causes the delay of spermatogenesis and vitellogenesis in crustaceans.

DOI： 10.1007/s10646-008-0228-7

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

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.

DOI： 10.1095/biolreprod.108.070052

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC FRANCAISE D ICHTYOLOGIE  

The effects of Ph, Mo, As and Rb on spermatogenesis were investigated by using testicular organ culture of Japanese eel (Anguilla japonica). Treatment with these trace elements together with 11-ketotestosterone (KT) significantly inhibited KT-induced germ cells proliferation, suggesting that Mo, Rb, Ph and As directly interrupt KT-induced spermatogenesis. We also investigated the effects of As on in vitro KT synthesis in testis. KT production induced by human chorionic gonadotropin (hCG) was inhibited by low doses of As. This result suggested that As also may directly inhibit KT production.

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

To stimate the influence of water contaminants on fish reproduction in the Mekong Delta area, we sampled cultivated male catfish (Pangasianodon hypophthalmus), investigated testicular development, and measured persistent organic pollutants (POPs) and trace element levels in muscle and liver, respectively. Various testes sizes were observed although sampling took place during a short period. Histological analysis revealed that all developmental stages of germ cells were observed in catfish with large testis, whereas only necrotic spermatogonia but no other germ cells were observed in catfish with small testis. In small testis, furthermore, vacuolization and hypertrophy of Sertoli cells were observed. Measurement of POPs in muscle and trace elements in liver demonstrated that there were negative correlations between GSI and the concentrations of Pb, Mo, Rb and As. To clarify possible direct effects of Pb, Mo, Rb and As on spermatogenesis in fish, we investigated the effects of these trace elements on spermatogenesis using in vitro testicular organ culture of Japanese eel (Anguilla japonica). Treatment with each of the trace elements alone did not affect spermatogenesis. However, treatment with 10(-7) M of Pb, 10(-5) and 10(-4) M of Mo, 10(-5) -10(-3) M of Rb or 10(-5) M of As inhibited the spermatogenesis induced by 11-ketotestosterone (11 KT). Furthermore, treatment with 10(-4) M of As in combination with 11KT caused necrosis of testicular fragments. Taken together, these results are consistent with the hypothesis that Ph, Mo, Rb and As can exert inhibitory effects on spermatogenesis in catfish inhabiting the Mekong Delta area. (C) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.aquatox.2007.03.010

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ENDOCRINE SOC  

In fish spermatogenesis, the main action of progestins is generally regarded as the induction of sperm maturation. Our previous in vitro study demonstrated that a progestin, 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP), induced the initiation of meiosis in spermatogenesis in the Japanese eel (Anguilla japonica). In the present study, to elucidate the molecular mechanisms underlying the action of DHP, we attempted to clone cDNAs encoding genes whose expression was induced by DHP in eel testis, using cDNA subtraction. One of the cDNAs we isolated encodes eel 11 beta-hydroxysteroid dehydrogenase short form (e11 beta-HSDsf), and Northern blot and RT-PCR analysis showed that transcripts of e11 beta-HSDsf in testis were induced by DHP. The recombinant e11 beta-HSDsf had 11 beta-dehydrogenase activity, metabolizing cortisol to cortisone, and 11 beta-hydroxytestosterone to 11-ketotestosterone (11-KT). In vitro experiments revealed that eel immature testis had 11 beta-dehydrogenase activity, and DHP treatment enhanced the activity. To understand the role of 11 beta-HSD in spermatogenesis, we examined the direct effects of cortisol on eel spermatogenesis using an organ culture system. Cortisol induced DNA replication in spermatogonia and enhanced the spermatogonial proliferation induced by 11-KT. However, excess cortisol inhibited proliferation. In addition, 11-KT production was induced in testicular fragments incubated with cortisol. These results suggest that optimal levels of cortisol induced spermatogonial mitosis by increasing 11-KT production. Furthermore, two possible roles of DHP on spermatogenesis, via the up-regulation of 11 beta-HSD expression, are suggested: positive feedback control of 11-KT production and the modulation of cortisol levels to protect testes from excess circulating cortisol.

DOI： 10.1210/en.2006-0391

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

In order to investigate the influence of estrogen and androgen on reproductive activities of male teleosts, male red sea bream were implanted with silicone capsules containing estradiol-17 beta (E2), testosterone (T) or 11-ketotestosterone (11-KT) in immature and early spermatogenic stages. One month after implantation of either E2 or T, the gonadosomatic index decreased in accordance with testicular regression in both stages. Implantation of E2 decreased circulating 11-KT levels but did not affect gonadotropin (GTH) subunits, follicle stimulating hormone-beta (FSH beta), luteinizing hormone-beta (LH beta), a glycoprotein subunit (alpha GSU) gene expression, and serum LH levels in both stages. Alternatively, T decreased serum 11-KT and LH levels, and FSH beta and LH beta mRNA levels in the early spermatogenic stage but not in the immature stage. These results suggest E2 may directly inhibit testicular development through the suppression of 11-KT production. Meanwhile, T may decrease serum 11-KT levels through the suppression of FSH and LH secretion, resulting to inhibition of testicular development in the early spermatogenic stage. Treatment with 11-KT did not affect the testis in either stage, whereas 11-KT increased LH beta and alpha GSU mRNA levels in immature, and decreased FSH beta mRNA levels in the early spermatogenic stage. These results suggest that 11-KT may have different effects on GTH subunit gene expression in each reproductive stage.

DOI： 10.1111/j.1444-2906.2006.01225.x

Web of Science

researchmap

Language：English   Publisher：ZOOLOGICAL SOC JAPAN  

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

The duality of gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), has been confirmed in most teleost species, but very little is known about their biological functions. To elucidate the physiological roles of FSH and LH in fish reproduction, the expression profiles of GTH subunit genes during gonadal development were analyzed in both male and female red seabream. Furthermore, in vitro studies were carried out to examine the effects of GTHs on steroid hormone production and cytochrome P450 aromatase (P450arom) expression in red seabream gonads. In both sexes, LHβ mRNA was maintained at high levels from the early gametogenesis until spawning season, and declined with gonadal regression. Interestingly, FSHβ mRNA levels in males increased in parallel with testicular development, whereas those in female were remained low throughout oocyte development. From in vitro studies using purified red seabream FSH and LH, both GTHs had a similar potency in stimulating 11-ketotestosterone production by testicular slices, while the biological activity of FSH was much lower than that of LH in stimulating production of estradiol-17β by vitellogenic follicles. Moreover, expression of P450arom mRNA was induced by LH, but not FSH, in ovarian follicles in vitro. FSH was also ineffective in inducing maturational competence and final oocyte maturation. These results suggest that, unlike salmonids, FSH may play an important role during gametogenesis in male, but not female, red seabream, whereas LH may be involved in regulation of both early and late gametogenesis in both sexes. © 2004 Kluwer Academic Publishers.

DOI： 10.1023/B:FISH.0000030480.97947.ba

Scopus

researchmap

Publishing type：Research paper (scientific journal)  

The steroid synthesis pathway in the ovarian follicles of the red seabream during final oocyte maturation (FOM) was investigated by incubating intact follicles with different radioactively labeled steroid precursors. During FOM, the steroidogenic shift from estradiol-17β to 20β-hydroxylated progestin production occurred mainly due to a combination of inactivation of C17,20-lyase and activation of 20β-hydroxysteroid dehydrogenase. Of the steroids produced, 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) exhibited the greatest effect on germinal vesicle breakdown (GVBD) in vitro. 17,20β-P was further converted to its 5β-reduced form, 17,20β-dihydroxy-5β-pregnan-3-one (17,20β-P-5β), which had lower GVBD activity, suggesting that 5β-reduction plays a role in the inactivation of the maturation-inducing ability of 17,20β-P. In contrast, no 5β-reduced metabolite of 20β-S was found. Serum levels of 17,20β-P and 20β-S, measured by ELISA, showed that circulating levels of both progestins increased during FOM, and 20β-S levels were approximately twice as high as 17,20β-P levels. This study clarified the complete steroidogenesis pathway during FOM in red seabream ovarian follicles and showed that two 20β-hydroxylated progestins, 17,20β-P and 20β-S, act as maturation-inducing hormones in this species. The catabolites of these two progestins and their physiological roles in reproduction are also discussed. © 2002 Elsevier Science Inc. All rights reserved.

DOI： 10.1016/S1096-4959(02)00106-9

Scopus

PubMed

researchmap

Publishing type：Research paper (scientific journal)  

The red seabream Pagrus major is a useful experimental fish for studying the endocrine control of oogenesis in teleosts. This study investigated the steroidogenic pathway for estradiol-17β (E2) biosynthesis in the ovarian follicles of red seabream. Intact follicles were isolated during vitellogenesis and incubated in vitro with different radiolabeled steroid precursors. When 17-hydroxyprogesterone (17-P), dehydroepiandrosterone (DHEA), or androstenedione (AD) were used as precursors, both testosterone (T) and estrone (E1) were synthesized by follicles, leading to estradiol-17β (E2) production. Serum steroid levels measured by enzyme-linked immunosorbent assay showed that T, E1, and E2 were present in the circulation at levels ranging from 1 ng/mL to 2 ng/mL throughout the day during the spawning season. In vitro conversion of E1 into E2, however, was 15.8-fold greater than T conversion into E2, suggesting that E2 is synthesized mainly via E1 rather than T. The results showed that E2 was synthesized from pregnenolone via 17-hydroxypregnenolone, DHEA, AD, and E1. Thus, the study demonstrated the complete steroidogenic E2 synthesis pathway in the ovarian follicles of red seabream, and revealed that E1 is the major precursor of E2.

DOI： 10.1046/j.1444-2906.2002.00477.x

Scopus

researchmap

Language：English   Publisher：SPRINGER TOKYO  

DOI： 10.2331/fishsci.68.sup1_953

Web of Science

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：English   Presentation type：Oral presentation (general)  

researchmap

Language：English   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Poster presentation  

researchmap

Language：English   Presentation type：Oral presentation (general)  

researchmap