Effects of Tributyltin-Contaminated Aquatic Environments and Remediated Water on Early Development of Sea Urchin (Hemisentrotus pulcherrimus).

In this study, gametotoxicity and embryotoxicity experiments were performed using Hemicentrotus pulcherrimus to investigate the toxic effects of tributyltin (TBT). The effects of TBT on fertilization and embryogenesis were assessed at various concentrations (0, 0.02, 0.05, 0.09, 0.16, 0.43, 0.73, 4.68, and 9.22 ppb). The fertilization rates decreased in a concentration-dependent manner, with significant reduction following treatment with TBT at 0.05 ppb. Embryos exhibited developmental impairment after TBT exposure at each tested concentration. The frequency of developmental inhibition delay that treatment with TBT delayed embryonic development in a dose-dependent manner, with 100% of embryos exhibiting developmental impairment at 4.68 ppb. During developmental recovery tests, embryos cultured in fresh media without TBT showed advanced embryonic development. Although the observed normal development after transferring the developmentally delayed embryos to fresh media without TBT offers prospects for the restoration of contaminated environments, embryonic development remained incomplete. These results suggest that TBT adversely affects the early embryonic development of H. pulcherrimus.

Effects of nonylphenol on early embryonic development, pigmentation and 3,5,3'-triiodothyronine-induced metamorphosis in Bombina orientalis (Amphibia: Anura).

Nonylphenol (NP) is an estrogenic endocrine disruptor in many aquatic species. In an effort to highlight the developmental toxicity of NP in amphibians, we examined the effects of NP on the embryonic survival, tadpole growth, melanophore development and metamorphosis of a native Korean amphibian species, Bombina orientalis (Anura). When treated to fertilized eggs, 1 µM NP significantly decreased embryonic survival at 48 h post fertilization (p.f.), suggesting that 1 µM NP can exert systemic toxicity in B. orientalis embryos. In the surviving embryos, there were no significant differences in malformation rates between NP-treated embryos and controls at 240 h p.f., suggesting no or low teratogenicity of NP in B. orientalis embryos. Below LC(50) NP significantly decreased body growth and development of melanophores at 0.1 µM, suggesting that NP far below the LC(50) targets multiple developmental events in tadpoles of this frog species. In metamorphosis assay using the premetamorphic tadpoles (corresponding to Nieuwkoop Faber stage 53 in Xenopus laevis) exogenous 3,5,3'-triiodothyronine (T3)-induced tail resorption was significantly decreased by 1 µM NP. However, NP (0.1 and 1 µM)-only treatment did not affected total body T3 and T4 levels, suggesting that NP at tested concentrations inhibits thyroid hormones action but not the synthesis of hormones during metamorphosis.

Bisphenol A modulates expression of gonadotropin subunit genes in the hermaphroditic fish, Kryptolebias marmoratus.

Pituitary gonadotropins (GTHs), follicle stimulating hormone beta (FSH-beta), and luteinizing hormone beta (LH-beta) are the key hormones in the hypothalamus-pituitary-gonad (HPG) axis, and form the heterodimers between a common alpha subunit (gonadotropin-alpha) and FSH-beta and/or LH-beta. To obtain a better understanding on the modulation of gonadotropin subunit genes expression upon bisphenol A (BPA) exposure in hermaphroditic fish, we studied differential regulation of gonadotropin subunit genes from Kryptolebias marmoratus after the exposure of several EDCs. Expression profiles of these three genes when using quantitative real-time RT-PCR revealed that brain/pituitary tissues were highly expressed in these genes compared to other tissues. At different developmental stages, expression of those genes dramatically increased over the course of development but showed a decrease in expression at the secondary male (showing atresia) stage. When adult fish were exposed to BPA (600 microg/L for 96 h), a significant upregulation of these three genes was observed in the brain/pituitary. A time course study also revealed the increased expression of gonadotropin subunit genes over 12 h with a more pronounced effect on the expression of FSH-beta and LH-beta genes, indicating that both genes were associated with the BPA exposure on the transcriptional regulation. This is the first report of gonadotropin subunit genes from K. marmoratus, with particular emphasis on the modulation of their expressions by EDCs. In addition, these findings suggest that EDCs modulate the expression of gonadotropin subunit genes and would act as potential biomarkers upon EDCs exposure.

Effect of carbaryl on survival and development in Bombina orientalis (Boulenger) embryos.

Bombina orientalis is one of the most common amphibians in the world and comprise a large proportion of their total number in Korea. B. orientalis, spawns in the farming regions at Spring when the massive application of agricultural chemicals occurs. Carbaryl, carbamate chemical is a slightly to highly toxic insecticide inhibiting acetylcholinesterase. The embryotoxicity and teratogenic effects of carbaryl on B. orientalis embryos were investigated at 5, 10, 50 and 100 muM. The survival rates of embryos at 312 h post fertilization were decreased with concentration dependent manner. Exposure to carbaryl produced 4 types of severe external abnormalities such as bent trunk, thick-set body, bent tail and ventral blister. At 5 muM carbaryl, a dose of no observed effect on embryonic survival, developmental abnormalities were significantly increased. The developmental abnormalities showed in order of frequency with bent trunk, thick-set body, bent tail and ventral blister. This result suggests that carbaryl is detrimental for embryonic survival and teratogenic by causing the axial skeletal defects in B. orientalis embryos.

Endocrine disruptors modulate expression of hepatic choriogenin genes in the hermaphroditic fish, Kryptolebias marmoratus.

Choriogenins (Chgs) are precursors of inner layer of egg envelope that are synthesized in fish liver in response to estrogens. Therefore, study of their expression serves as biomarker of exposure to endocrine disrupting chemicals (EDCs). The self-fertilizing fish, Kryptolebias marmoratus has been established as a model species for testing the action of EDCs. To use this fish as a model for assessing estrogenic activity of EDCs on Chg expression, two K. marmoratus choreogenin genes, Km-ChgH and Km-ChgL were cloned and their expression was analyzed in different tissues and in developmental stages by real-time RT-PCR. Expression levels of liver mRNA were compared between hermaphrodites and secondary males after exposure to EDCs. Km-ChgH and Km-ChgL genes that were predominantly expressed in liver contained zona pellucida (ZP) domains. During embryonic development, low expression of mRNA was observed at stage 1 (2 dpf) that reached highest level at stage 4 (12 dpf) or stage 5 (5 h post hatching). The expression of Km-Chg mRNAs was highly increased in liver exposed to natural estrogen, 17alpha-estradiol (E2) as well as EDCs such as bisphenol A and 4-n-nonylphenol in both the gender types. Another EDC, 4-tert-octylphenol, showed modulatory effect only on Km-ChgH in hermaphrodites. Tamoxifen, an antagonist of the estrogen receptor showed no effect on expression of Chg genes in either of the gender types of K. marmoratus. These findings indicate that Km-Chg genes would be associated with estrogen and measurement of their expression would serve as a surrogate biomarker of exposure to environmental EDCs.

Effects of endosulfan on survival and development of Bombina orientalis (Boulenger) embryos.

The developmental toxicity of endosulfan was examined in the anuran Bombina orientalis embryos. Survival rates of embryos following 50 microM endosulfan treatment was significantly lower than vehicle control at 96 h onward. When the embryos develop to the tail fin circulation stage, embryonic survival was significantly decreased by 10 microM endosulfan treatment. Surviving embryos showed various developmental abnormalities including tail dysplasia at 50 microM. By hampering the embryonic development endosulfan may cause the decline in the natural populations of this frog species breeding on farmland and in the surrounding aquatic environment.

Effects of aroclor 1254 on the expression of the KAP3 gene and reproductive function in rats.

The present study investigated the effects of aroclor 1254 (A1254) on the expression of the kinesin superfamily associated protein 3 (KAP3) gene in F1 rat brain during brain sexual differentiation and puberty. In addition, the effects of A1254 on reproductive function were examined. The KAP3 gene is involved in the neurogenesis and synaptogenesis of sexual differentiation in rats and also during puberty. In the present study, pregnant Sprague-Dawley rats each received a daily dose of A1254 (0, 10, 50 mg kg(-1)) dissolved in 1.0 mL corn oil by gavage, from gestational Day (GD) 8 to postnatal Day (PD) 21. The mRNA levels of the KAP3 gene in hypothalamic tissues were analysed by northern blot hybridisation during the critical periods of brain sexual differentiation (GD18 and PD5) and puberty (PD28). Variables affecting reproduction in F1 female rats, such as vaginal opening (VO), vaginal oestrus (VE) and oestrous cyclicity, were recorded. Depending on the sex and A1254 exposure (control or 50 mg kg(-1) day(-1)), F1 rats were divided into three mating groups, namely control male-control female, control male-A1254-treated female and A1254-treated male-control female. During the critical periods of brain sexual differentiation (GD18, PD5) and puberty (PD28), KAP3 mRNA levels were significantly reduced in A1254-treated fetal and pubertal rat brains relative to those of control groups. In A1254-treated F1 female rats, VO and VE were delayed, the percentage of irregular oestrous cycles was increased and the duration of the oestrous cycle was extended in a dose-dependent manner compared with control groups. Treatment with a high dose of A1254 significantly impaired the reproductive function of both male and female F1 rats, including mating and pregnancy indices and the number of live fetuses. These data suggest that A1254 disrupts transcriptional regulation of the KAP3 gene in fetal and pubertal rat brains and that these effects may be related to A1254-induced abnormal brain sexual differentiation and lowered reproductive function in F1 rats.

CDNA cloning and expression of ATP synthase subunit b from the fire-bellied frog Bombina orientalis (Anura: Discoglossidae).

We cloned the Bombina orientalis adenosine triphosphate (ATP) synthase subunit b gene from a B. orientalis oviduct cDNA library. The transcript was 997 bp long and encoded 250 amino acid residues. It showed high similarity to amphibian (84-85%), mammalian (56-62%) and invertebrate (46-50%) sequences. In phylogenetic analyses, it clustered with other amphibian sequences. This gene was highly expressed in brain, intestine and oviduct but not in muscle and liver. In this paper, we report the basic characteristics of B. orientalis ATP synthase subunit b gene.

Gene expression analysis of the pro-oestrous-stage rat uterus reveals neuroligin 2 as a novel steroid-regulated gene.

In the present study, differential gene expression in the uteri of ovariectomised (OVX) and pro-oestrous rats (OVX v. pro-oestrus pair) was investigated using cDNA expression array analysis. Differential uterine gene expression in OVX rats and progesterone (P(4))-injected OVX rats (OVX v. OVX + P(4) pair) was also examined. The uterine gene expression profiles of these two sets of animals were also compared for the effects of P(4) treatment. RNA samples were extracted from uterine tissues and reverse transcribed in the presence of [alpha(32)P]-dATP. Membrane sets of rat arrays were hybridised with cDNA probe sets. Northern blot analysis was used to validate the relative gene expression patterns obtained from the cDNA array. Of the 1176 cDNAs examined, 23 genes showed significant (>two-fold) changes in expression in the OVX v. pro-oestrus pair. Twenty of these genes were upregulated during pro-oestrus compared with their expression in the OVX rat uterus. In the OVX v. OVX + P(4) pair, 22 genes showed significant (>two-fold) changes in gene expression. Twenty of these genes were upregulated in the OVX + P(4) animals. The genes for nuclear factor I-XI, afadin, neuroligin 2, semaphorin Z, calpain 4, cyclase-associated protein homologue, thymosin beta-4X and p8 were significantly upregulated in the uteri of the pro-oestrus and OVX + P(4) rats of both experimental pairs compared with the OVX rat uteri. These genes appear to be under the control of P(4). One of the most interesting findings of the present study is the unexpected and marked expression of the neuroligin 2 gene in the rat uterus. This gene is expressed at high levels in the central nervous system and acts as a nerve cell adhesion factor. According to Northern blot analysis, neuroligin 2 gene expression was higher during the pro-oestrus and metoestrus stages than during the oestrus and dioestrus stages of the oestrous cycle. In addition, neuroligin 2 mRNA levels were increased by both 17beta-oestradiol (E(2)) and P(4), although P(4) administration upregulated gene expression to a greater extent than injection of E(2). These results indicate that neuroligin 2 gene expression in the rat uterus is under the control of both E(2) and P(4), which are secreted periodically during the oestrous cycle.

Circadian expression of Mel1a and PL-II genes in placenta: effects of melatonin on the PL-II gene expression in the rat placenta.

In the mammal, melatonin regulates the seasonal and/or circadian rhythm of PRL levels. Since several members of the PRL gene family are expressed during late pregnancy, we investigated the relationship between the expression of placental lactogen (PL)-II-one member of the PRL family- and melatonin, as well as the placental expression of one of the receptors for melatonin, melatonin receptor 1a (Mel(1a())). Herein we provide the first demonstration that Mel(1a) is not only expressed in the rat placenta, but that it is spatially and temporally regulated throughout late pregnancy. In situ hybridization and Northern blot analyses show that Mel(1a) mRNA is localized in the rat placenta on gestational day 19, and is mainly restricted to the spongiotrophoblast and trophoblast giant cells. Interestingly, the junctional zone of the placenta at this time showed the strongest gene expression when the tissue was obtained at 16:00 h (daytime) and showed the least expression when it was obtained at 04:00 h (night-time). In contrast, the labyrinth zone showed the strongest expression in tissue obtained at night and showed the least expression in tissue obtained during the day. PL-II gene expression also exhibited a circadian rhythm but the direction of the fluctuation was exactly opposite to that of the Mel(1a) gene, such that at night the junctional zone had the strongest expression, while the labyrinth zone had the weakest. In vitro treatment of placental tissue with an melatonin agonist, chloromelatonin, greatly decreased PL-II mRNA levels. That Mel(1a) plays a regulatory role in the expression of PL-II in the late-pregnancy rat placenta is strongly suggested by the pattern of its own spatial and temporal expression.