Search

The brightness and color of pearls varies among different pearl-producing shellfish and have been a source of human fascination since ancient times. When produced through cultivation, the characteristics and quality of a pearl depend on the kind of shellfish used and also the transplanted mantle graft. This suggests that the Akoya pearl oyster, which is generally used in Japan for pearl culturing, can produce different kinds of pearl through the use of mantles from different species of shellfish. However, a transplanted heterogeneous mantle would be rejected by the immune system of the Akoya oyster. We have therefore developed a new method to suppress the Akoya immune system that archives immune tolerance to other shellfish. It is generally known that small quantities of antigens can be used to produce archived immunological tolerance in a clinical setting. We successfully suppressed the Akoya pearl oyster immune response against a Mabé pearl oyster graft through repeat injections of mantle homogenates. We then transplanted a Mabé pearl oyster mantle graft into the immunologically tolerant Akoya pearl oyster and obtained a Mabé pearl from an Akoya pearl oyster. Our new technique thus makes the production of novel and different pearls in the Akoya possible. We believe that this has significant future potential for the advancement of the pearl industry.

It has been demonstrated that taurine has various physiological functions in the body. We demonstrated that taurine is abundant in the serum, liver, muscle and testis of the Japanese eel (Anguilla japonica). In the eel testis, taurine is found mainly in spermatogonia and is weakly expressed also in the Sertoli cells. We have further found in the eel testis that taurine is actively accumulated via the sodium/chloride-dependent taurine transporter (TauT; SLC6A6), which is expressed in germ cells. In our current study, the effects of taurine on the anti-oxidant response were examined. Taurine was found to promote the total superoxide dismutase (SOD) activity in the testis. Moreover, our results indicate that taurine does not affect the mRNA levels of copper-zinc (Cu/Zn) SOD or manganese SOD, but promotes the translation of Cu/Zn SOD. Overall, our present data suggest that taurine may modulate Cu/Zn SOD at the translational level and thereby may play an important role in the protection of germ cells from oxidative stress.

Oxidative stress has been implicated in pathogenesis of many diseases, but few studies describe its influence on spermatogenesis. In this study, we analyzed the direct influence of hypoxanthine (Hx)-induced reactive oxygen species (ROS) on spermatogenesis in fish using the Japanese eel (Anguilla japonica) testicular organ culture system. Testicular fragments of eels were cultured in 0.1-100 μM Hx with or without 10 ng/ml 11-ketotestosterone (11-KT). Immunohistochemistry for 5-bromo-2-deoxyuridine showed that Hx treatment at a low dose (1 μM) already inhibits 11-KT-induced germ cell proliferation after culture. An in situ TUNEL assay and 8-hydroxy-2'-deoxyguanosine immunohistochemistry revealed an intense germ cell apoptosis and high oxidative DNA damage in testicular fragments cultured at the highest dose of Hx (100 μM) with 11-KT. A total superoxide dismutase (SOD) activity assay showed a decrease in SOD activity in testicular fragments cultured with 11-KT. These data suggest that ROS may directly inhibit spermatogenesis, and that decreased SOD activity renders proliferating spermatogonia susceptible to ROS, hence leading to apoptosis.

In teleost fish, the progestin 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) is an essential component of the spermatogenesis pathway. In a series of investigations on the mechanisms underlying progestin-stimulated spermatogenesis, we have found that DHP up-regulates the expression of cysteine dioxygenase1 (CDO1) in the Japanese eel testis. CDO1 is one of the enzymes involved in the taurine biosynthesis pathway. To evaluate whether taurine is synthesized in the eel testis, cysteine sulfinate decarboxylase (CSD), another enzyme involved in taurine synthesis, was isolated from this species. RT-PCR and in vitro eel testicular culture revealed that although CSD was also expressed in eel testis, neither DHP nor other sex steroids affect CSD mRNA expression in a similar manner to CDO1. Using an in vitro eel testicular culture system, we further investigated the effects of DHP on taurine synthesis in the eel testis. HPLC analysis showed that DHP treatment significantly increases the taurine levels in the eel testis. These results suggest that DHP promotes taurine synthesis via the up-regulation of CDO1 mRNA expression during eel spermatogenesis. Furthermore, we observed from our analysis that although taurine does not induce complete spermatogenesis, it promotes spermatogonial DNA synthesis and the expression of Spo11, a meiosis-specific marker. These data thus suggest that taurine augments the effects of sex steroids in the promotion of spermatogonial proliferation and/or meiosis and hence that taurine plays important roles in spermatogenesis.

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-2'-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.

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 microM 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 microM arsenic and hCG for 18 h. A 0.1 microM (7 microg/l) dose of arsenic which is lower than the World Health Organization drinking water quality guideline of 10 microg/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 microM), implying an inhibition of 3beta-hydroxysteroid dehydrogenase activity. In situ TUNEL assays indicated that germ cells undergo apoptosis at the highest dose of arsenic (100 microM). 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 microM 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.

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, 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP), the initiator of meiosis in fish, and estradiol-17beta (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.

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 microg/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 microg/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.