Folic acid protects against arsenic-mediated embryo toxicity by up-regulating the expression of Dvr1.

As a nutritional factor, folic acid can prevent cardiac and neural defects during embryo development. Our previous study showed that arsenic impairs embryo development by down-regulating Dvr1/GDF1 expression in zebrafish. Here, we investigated whether folic acid could protect against arsenic-mediated embryo toxicity. We found that folic acid supplementation increases hatching and survival rates, decreases malformation rate and ameliorates abnormal cardiac and neural development of zebrafish embryos exposed to arsenite. Both real-time PCR analysis and whole in-mount hybridization showed that folic acid significantly rescued the decrease in Dvr1 expression caused by arsenite. Subsequently, our data demonstrated that arsenite significantly decreased cell viability and GDF1 mRNA and protein levels in HEK293ET cells, while folic acid reversed these effects. Folic acid attenuated the increase in subcellular reactive oxygen species (ROS) levels and oxidative adaptor p66Shc protein expression in parallel with the changes in GDF1 expression and cell viability. P66Shc knockdown significantly inhibited the production of ROS and the down-regulation of GDF1 induced by arsenite. Our data demonstrated that folic acid supplementation protected against arsenic-mediated embryo toxicity by up-regulating the expression of Dvr1/GDF1, and folic acid enhanced the expression of GDF1 by decreasing p66Shc expression and subcellular ROS levels.

Taurine improves the spatial learning and memory ability impaired by sub-chronic manganese exposure.

BACKGROUND: Excessive manganese exposure induced cognitive deficit. Several lines of evidence have demonstrated that taurine improves cognitive impairment induced by numerous neurotoxins. However, the role of taurine on manganese-induced damages in learning and memory is still elusive. This goal of this study was to investigate the beneficial effect of taurine on learning and memory capacity impairment by manganese exposure in an animal model. RESULTS: The escape latency in the Morris Water Maze test was significantly longer in the rats injected with manganese than that in the rats received both taurine and manganese. Similarly, the probe trial showed that the annulus crossings were significantly greater in the taurine plus manganese treated rats than those in the manganese-treated rats. However, the blood level of manganese was not altered by the taurine treatment. Interestingly, the exposure of manganese led to a significant increase in the acetylcholinesterase activity and an evidently decrease in the choline acetyltransferase activity, which were partially restored by the addition of taurine. Additionally, we identified 9 differentially expressed proteins between the rat hippocampus treated by manganese and the control or the manganese plus taurine in the proteomic analysis using the 2-dimensional gel electrophoresis followed by the tandem mass spectrometry (MS/MS). Most of these proteins play a role in energy metabolism, oxidative stress, inflammation, and neuron synapse. CONCLUSIONS: In summary, taurine restores the activity of AChE and ChAT, which are critical for the regulation of acetylcholine. We have identified seven differentially expressed proteins specifically induced by manganese and two proteins induced by taurine from the rat hippocampus. Our results support that taurine improves the impaired learning and memory ability caused by excessive exposure of manganese.

Dexamethasone altered steroidogenesis and changed redox status of granulosa cells.

Glucocorticoids have been widely used in clinical application for anti-inflammatory and immunosuppressive function. Previous study reported that glucocorticoids adversely affect the reproductive system and can directly act on ovary. Here, we found that progesterone production induced by dexamethasone requiring activation of caspase-3 which may mediate differentiation and apoptosis of granulosa cells. Further study displayed that cellular glutathione level was increased and reactive oxygen species was decreased accompanied with unchanged mitochondrial membrane potential which may contribute to the maintenance of steroidogenesis in granulosa cells treated with dexamethasone. Dexamethasone also augmented the level of anti-Müllerian hormone secreted by preovulatory granulosa cells which indicated that dexamethasone may promote preantral follicles development.

Embryonic developmental toxicity of selenite in zebrafish (Danio rerio) and prevention with folic acid.

Selenium (Se) is an essential micronutrient, but also a potential toxin, which may be absorbed in excess. Relatively little is known about selenium embryotoxicity in zebrafish. In this study, we evaluated the effect of selenite exposure in zebrafish embryos. Selenite treatment decreased survival and resulted in abnormal development in a dose- and time-dependent manner. We observed irregular growth of neurons in selenite treated embryos, characterized by the absence of neurons in the brain, trunk and tail. Selenite exposure also induced defects in heart function, such as bradycardia and cardiac dysplasia with irregular and smaller chamber shape. In addition, selenite exposure caused ectopic cell proliferation, apoptosis, and a change in the pattern of DNA methylation. Our results suggested that supplementation with folic acid (FA) ameliorated the cardiac and neural defects in selenite-treated embryos. In conclusion, we demonstrated that selenite exposure caused cardiac and neural defects in zebrafish embryos and that folic acid protected against this embryotoxicity. It will give insight into the risk assessment and prevention of Se-mediated embryotoxicity.

A novel mutation impairing the tertiary structure and stability of γC-crystallin (CRYGC) leads to cataract formation in humans and zebrafish lens.

Congenital cataract is one of the leading causes of human blindness. In this study, we identified a novel, heterozygous c.385G

Arsenic induced progesterone production in a caspase-3-dependent manner and changed redox status in preovulatory granulosa cells.

Arsenic contamination is a principal environmental health threat throughout the world. However, little is known about the effect of arsenic on steroidogenesis in granulosa cells (GCs). We found that the treatment of preovulatory GCs with arsenite stimulated progesterone production. A significant increase in serum level of progesterone was observed in female Sprague-Dawley rats following arsenite treatment at a dose of 10 mg/L/rat/day for 7 days. Further experiments demonstrated that arsenite treatment did not change the level of intracellular cyclic AMP (cAMP) or phosphorylated ERK1/2 in preovulatory GCs; however, progesterone production was significantly decreased when cAMP-dependent protein kinase (PKA) or ERK1/2 pathway was inhibited. This implied that the effect of arsenite on progesterone production may require cAMP/PKA and ERK1/2 signaling but not depend on them. Furthermore, we found that arsenite decreased intracellular reactive oxygen species (ROS) but increased the antioxidant glutathione (GSH) levels and mitochondrial membrane potential (ΔΨm) in parallel to the changes in progesterone production. Progesterone antagonist blocked the arsenic-stimulated increase of GSH levels. Arsenite treatment induced caspase-3 activation, although no apoptosis was observed. Inhibition of caspase-3 activity significantly decreased progesterone production stimulated by arsenite or follicle-stimulating hormone (FSH). GSH depletion with buthionine sulfoximine led to cell apoptosis in response to arsenite treatment. Collectively, this study demonstrated for the first time that arsenite stimulates progesterone production through cleaved/active caspase-3-dependent pathway, and the increase of GSH level promoted by progesterone production may protect GCs against apoptosis and maintain the steroidogenesis of GCs in response to arsenite treatment.

[Role of extracellular signal-regulated protein kinase 5 in the biosynthesis of follicle-stimulating hormone-stimulated progesterone in primary granulosa cells].

OBJECTIVE: To study the role of extracellular signal-regulated protein kinase 5 (ERK5) during the biosynthesis of follicle-stimulating hormone (FSH)-mediated progesterone in primary granulosa cells. METHODS: The expressions of phosphorylated and general forms of ERKS in primary granulosa cells after the treatment of FSH were detected by Western blot analysis. The subcellular localization of ERK5 was observed by confocal microscopy. The effect of ERK5 on FSH-mediated progesterone biosynthesis in primary granulosa cells was analyzed using recombinant adenovirus vectors. RESULTS: ERK5 activation was induced by FSH in a time-dependent manner in primary cultured granulosa cells, although the general ERK5 protein level decreased also in a time-dependent manner. The treatment of FSH showed no remarkable effect on the subcellular distribution of endogenous ERK5, which was mainly in the cytoplasm of granulosa cells. The co-infection of Ad-caMEK5 and Ad-wtERK5 increased the progesterone production and StAR expression in primary cultured granulosa cells, whereas inhibition of ERK5 activation suppressed the FSH-stimulated progesterone production. CONCLUSION: ERK5 may stimulate FSH-mediated progesterone production in primary cultured granulosa cells.

Follicle-stimulating hormone regulation of microRNA expression on progesterone production in cultured rat granulosa cells.

MicroRNAs (miRNAs) regulate gene expression post-transcriptionally by interacting with the 3' untranslated regions of their target mRNAs. Previously, miRNAs have been shown to regulate genes involved in cell growth, apoptosis, and differentiation, but their role in ovarian granulosa cell follicle-stimulating hormone (FSH)-stimulated steroidogenesis is unclear. Here we show that expression of 31 miRNAs is altered during FSH-mediated progesterone secretion of cultured granulosa cells. Specifically, 12 h after FSH treatment, miRNAs mir-29a and mir-30d were significantly down-regulated. However, their expression increased after 48 h. Bioinformatic analysis used to predict potential targets of mir-29a and mir-30d revealed a wide array of potential mRNA target genes, including those encoding genes involved in multiple signaling pathways. Taken together, our results pointed to a novel mechanism for the pleiotropic effects of FSH.

Polycomb group proteins are essential for spinal cord development.

Birth defects are the leading cause of infantile mortality, followed by neural tube defects (NTD) and congenital heart defects. Spina bifida and anencephaly are among the most common forms of NTD. NTD etiologies are complex, and are associated with both genetic and environmental factors. Polycomb group proteins are essential for vertebrate development; therefore, the purpose of this study was to determine the role of PcGs in spinal cord morphogenesis in normal and all-trans-retinoic acid (RA)-treated fetal rat models of spina bifida. Pregnant rats were gavage-fed RA, resulting in fetal NTD, and embryos were obtained on day 15.5, 17.5, and 19.5. Western blot and immunohistochemistry were used to reveal PcGs expression in the normal and RA-treated E15.5-19.5 rat sacral cords. Western blot and immunohistochemistry revealed decreased EED, RNF2, SUZ12, and H3K27me3 expression in the normal, E15.5-19.5, rat sacral cords. In addition, the spinal cord of RA-treated rats during embryonic development exhibited altered PcGs protein expression. Administration of excess RA results in NTD. Our results suggest that the Polycomb proteins may be involved in spinal cord development.

Mouse microRNA-124 regulates the expression of Hes1 in P19 cells.

MicroRNAs (miRNAs) belong to a conserved class of small non-coding RNAs that are typically 18-25 nucleotides long. They are found in both animals and plants. These small RNAs can regulate gene expression at translational level by interacting with their target messenger RNAs, and they play an essential role in the development of plants and animals. To date, more than 200 miRNAs have been identified in mammals; however, their mRNA targets have not yet been identified. In this study, we demonstrate that the expression of Hes-1, which is a basic helix-loop-helix transcriptional repressor, is regulated by miRNA-124 in P19 cells. Reduction in the levels of miR-124 mediated by locked nucleic acids resulted in the accumulation of Hes-1 and hindered the retinoic acid-induced neuronal differentiation of P19 cells. Thus, our results indicate that miR-124 regulates the expression of Hes-1 at the post-transcriptional level and is involved in the retinoic acid-induced neuronal differentiation of P19 cells.

A network of miRNAs expressed in the ovary are regulated by FSH.

The process of folliculogenesis requires a tightly regulated series of gene expression that are a pre-requisite to the development of ovarian follicle. Among these genes, follicle-stimulating hormone (FSH) is notable for its dual role in development of follicles as well as proliferation and differentiation of granulosa cells. The post-transcriptional expression of these genes is under the control of microRNAs (miRNAs), a class of small, endogenous RNAs that negatively impact gene expression. This study was carried out to determine the role of several miRNAs including mir-143, let-7a, mir-125b, let-7b, let-7c, mir-21 in follicular development in the mouse. The expression of these RNAs was very low in primordial follicles but these became readily detectable in the granulosa cells of primary, secondary and antral follicles. We show that this expression of some miRNAs (mir-143, let-7a, mir-15b) is under negative control of FSH. Together, these findings suggest that FSH regulates folliculogenesis by a novel pathway of miRNAs.

[Effect of manganese on cytosolic free calcium concentration in cortical neurons].

OBJECTIVE: To investigate the change of free Ca(2+) in cytoplasma in the neurotoxicity of the manganese (Mn). METHODS: The cortical neurons were separated from the neonatal Wistar rats and cultured in vitro. The neurons were grouped as the Mn-treated groups and the untreated group. The neurons in the Mn-added groups were incubated in the culture media containing lower, medium and high dosage manganese chloride (MnCl(2 x 4) H2O) with the concentration at 0.2, 0.6, 1.0 mmol/L respectively. Meanwhile, neurons in control were cultured in the normal culture media. All treatments stopped 24 h later. Neurons were labeled Ca(2+) sensitive prober, Fluo-3/AM. The fluorescence intensity of Fluo-3 combined with Ca(2+) was examined by LSCM (Laser scanning confocal microscope) and was treated by the picture analysis technique. The intensity was equal to the free Ca(2+) concentrations in cytoplasma of neurons. RESULTS: MnCl(2) can induce free Ca(2+) overloaded in cytoplasma of neurons, but the increasing degree varied in MnCl(2) dosage. Cytoplasma Ca(2+) concentration in the moderate dosage The moderate dosage MnCl(2) group and the high dosage MnCl(2) group were significantly higher than that in the lower dosage MnCl(2) group and the control group (P < 0.05). CONCLUSION: The Ca(2+) overload is involved in the neurotoxicity of manganese, and a dosage response relationship is found between the manganese chloride dose and Ca(2+) overload in cortical neurons.