Identification of Hox genes and their expression profiles during embryonic development of the emerging model organism, Macrobrachium olfersii.

Hox genes encode transcription factors that specify the body segment identity during development, including crustaceans, such as amphipods and decapods, that possess a remarkable diversity of segments and specialized appendages. In amphipods, alterations of specialized appendages have been obtained using knockout experiment of Hox genes, which suggests that these genes are involved in the evolution of morphology within crustaceans. However, studies of Hox genes in crustaceans have been limited to a few species. Here, we identified the homeodomain of nine Hox genes: labial (lab), proboscipedia (pb), Deformed (Dfd), Sex combs reduced (Scr), fushi tarazu (ftz), Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abdA), and Abdominal-B (AbdB), and evaluated their expression by RT-qPCR and RT-PCR in the ovary, during embryonic development, and at the first larval stage (Zoea I) of the decapod Macrobrachium olfersii. The transcript levels of lab, Dfd, and ftz decreased and transcripts of pb, Scr, Antp, Ubx, abdA, and AbdB increased during embryonic development. Hox genes were expressed in mature ovaries and Zoea I larval stages, except Scr and ftz, respectively. In addition, isoforms of Dfd, Scr, Ubx, and abdA, which have been scarcely reported in crustaceans, were described. New partial sequences of 87 Hox genes from other crustaceans were identified from the GenBank database. Our results are interesting for future studies to determine the specific function of Hox genes and their isoforms in the freshwater prawn M. olfersii and to contribute to the understanding of the diversity and evolution of body plans and appendages in Crustaceans.

Ultraviolet B radiation affects epithelial cell morphology and ultrastructure in the hepatopancreas of the freshwater decapod Macrobrachium olfersii.

The hepatopancreas is the digestive organ of crustaceans, and plays important roles also in the synthesis and secretion of sexual hormones, immunological defenses and xenobiotic detoxification. Although the importance of this organ in crustaceans cannot be underestimated, the effects of ultraviolet B (UVB) radiation on hepatopancreas are poorly understood. Moreover, Macrobrachium prawns, have a transparent carapace, which make them more susceptible to UVB radiation, since their internal organs, such as hepatopancreas, are easily reached by solar radiation. Therefore, we aimed to evaluate UVB radiation toxicity on the morphology and morphometry of hepatopancreatic epithelial cells, and to investigate these UVB effects in subcellular compartments of the ecologically-important freshwater decapod, Macrobrachium olfersii. Hepatopancreas from the UVB-irradiated group showed a granular cytoplasm, with non-defined cell limits. Morphometric analyses revealed that the UVB-irradiated group exhibited a higher frequency of fibrillar (F-cell), resorptive (R-cell) and midget (M-cell), and decreased the blister-like (B-cell). It was also observed increased vacuole frequencies and increased F-, B- and R-cell volumes in the UVB-irradiated group. In addition, it was observed increased B-cell vacuolar volumes and decreased R-cell vacuolar volumes. Ultrastructural alterations occurred in subcellular compartments in F- and R-cells, e.g. loss of mitochondrial crests, morphologically compatible with mitochondrial fission, rough endoplasmic reticulum cisternae dilation, dilation of Golgi lamellar sacs, and increased vacuole and concentric membrane formation in the UVB-irradiated group. Our data showed that the hepatopancreas is an important target of UVB radiation, as demonstrated by a series of organ-specific morphological and morphometric impairments. Therefore, cell damage caused by UVB radiation can compromise metabolic functions in epithelial cells from the hepatopancreas, potentially affecting absorption, secretion and digestion processes, vitellogenin synthesis, immune responses and xenobiotic detoxification.

Exposure to homocysteine leads to cell cycle damage and reactive gliosis in the developing brain.

Studies that investigate the cellular effects of homocysteine (Hcy) on the differentiation of neural cells, and their involvement in establishment of cell layers in the developing brain are scarce. This study evaluated how Hcy affects the neural cell cycle and proteins involved in neuronal differentiation in the telencephalon and mesencephalon using the chicken embryo as a model. Embryos at embryonic day 2 (E2) received 20 µmol D-L Hcy/50 µl saline and analyzed at E6. The Hcy treatment induced an increase in the ventricular length of the telencephalon and also a reduction of the mantle layer thickness. We observed that Hcy induced impairments to the neural cell cycle and differentiation, which compromised the cell layers establishment in the developing brain. Hcy treatment also induced changes in gene and protein expression of astrocytes, characteristic of reactive gliosis. Our results point to new perspectives of evaluation of cellular targets of Hcy toxicity.

Impact of homocysteine on vasculogenic factors and bone formation in chicken embryos.

Developmental endochondral ossification requires constant blood supply, which is provided by the embryonic vascular network. High levels of homocysteine (Hcy) have vasculotoxic properties, but it remains unclear how Hcy disrupts blood vessel formation in endochondral ossification. Thus, we investigated the toxicity of Hcy on contents of vasculogenic factors (VEGF, VCAM-1, NOS3) and osteocalcin, using developing limbs as model. Chicken embryos were submitted to treatment with 20 µmol D-L Hcy at 12H&H and the analyses occur at 29H&H and 36H&H. We did not identify differences in the area of limb ossification in Hcy-treated (7.5 × 105 µm2 ± 3.9 × 104) and untreated embryos (7.6 × 105 µm2 ± 3.3 × 104) at 36H&H. In Hcy-treated embryos, we observed a significantly decrease of 46.8% at 29H&H and 26.0% at 36H&H in the number of VEGF-reactive cells. Also, treated embryos showed decrease of 98.7% in VCAM-1-reactive cells at 29H&H and 34.6% at 36H&H. The number of NOS3-reactive cells was reduced 54.0% at 29H&H and 91.5% at 36H&H, in the limbs of Hcy-treated embryos. Finally, in Hcy-treated embryos at 36H&H, we observed a reduction of 58.86% in the number of osteocalcin-reactive cells. Here, we demonstrated for the first time that the toxicity of Hcy is associated with a reduction in the contents of proteins involved in blood vessel formation and bone mineralization, which interferes with endochondral ossification of the limb during embryonic development. Graphical abstract.

microRNAs in Macrobrachium olfersii embryos: Identification, their biogenesis components and potential targets.

In embryonic development, microRNAs (miRNAs) regulate the complex gene expression associated with the complexity of embryogenesis. Today, few studies have been conducted on the identification of miRNAs and components of miRNA biogenesis on embryonic development in crustaceans, especially in prawns. In this context, the aim of this study was to identify in silico components of miRNA biogenesis, and miRNAs and potential target genes during embryonic development in the prawn Macrobrachium olfersii through small RNAs and transcriptome analyses. Using the miRDeep2 program, we identified 17 miRNA precursors in M. olfersii, which seven (miR-9, miR-10, miR-92, miR-125, miR-305, miR-1175, and miR-2788) were reported in the miRBase database, indicating high evolutionary conservation of these sequences among animals. The other 10 miRNAs of M. olfersii were novel miRNAs and only similar to Macrobrachium niponnense miRNAs, indicating genus-specific miRNAs. In addition, eight key components of miRNA biogenesis (DROSHA, PASHA/DGCR8, XPO5, RAN, DICER, TRBP2, AGO, and PIWI) were identified in M. olfersii embryos unigenes. In the annotation of miRNA targets, 516 genes were similar to known sequences in the GenBank database. Regarding the conserved miRNAs, we verified that they were differentially expressed during embryonic development in M. olfersii. In conclusion, this is the first study that identifies conserved and novel miRNAs in the prawn M. olfersii with some miRNA target genes involved in embryonic development. Our results will allow further studies on the function of these miRNAs and miRNA biogenesis components during embryonic development in M. olfersii and other prawns of commercial interest.

Low-concentration exposure to glyphosate-based herbicide modulates the complexes of the mitochondrial respiratory chain and induces mitochondrial hyperpolarization in the Danio rerio brain.

Glyphosate (N-phosphonomethyl-glycine) (GLY) is the active ingredient of the most used herbicides in the world. GLY is applied in formulated products known as glyphosate-based herbicides (GBH), which could induce effects that are not predicted by toxicity assays with pure GLY. This herbicide is classified as organophosphorus compound, which is known to induce neurotoxic effects. Although this compound is classified as non-neurotoxic by regulatory agencies, acute exposure to GBH causes neurological symptoms in humans. However, there is no consensus in relation to neurotoxic effects of GBH. Thus, the aim of this study was to investigate the neurotoxic effects of the GBH in the zebrafish Danio rerio, focusing on acute toxicity, the activity and transcript levels of mitochondrial respiratory chain complexes, mitochondrial membrane potential, reactive species (RS) formation, and behavioral repertoire. Adult zebrafish were exposed in vivo to three concentrations of GBH Scout®, which contained GLY in formulation (fGLY) (0.065, 1.0 and 10.0 mg L-1 fGLY) for 7 d, and an in vitro assay was performed using also pure GLY. Our results show that GBH induced in zebrafish brain a decrease in cell viability, inhibited mitochondrial complex enzymatic activity, modulated gene expression related to mitochondrial complexes, induced an increase in RS production, promoted hyperpolarization of mitochondrial membrane, and induced behavioral impairments. Together, our data contributes to the knowledge of the neurotoxic effects of GBH. Mitochondrial dysfunction has been recognized as a relevant cellular response that should not be disregarded. Moreover, this study pointed to the mitochondria as an important target of GBH.

Effect of UVB radiation exposure in the expression of genes and proteins related to apoptosis in freshwater prawn embryos.

Our previous studies showed that embryos of the freshwater prawn Macrobrachium olfersii exposed to ultraviolet B (UVB) radiation exhibited DNA damage, excessive ROS production, mitochondrial dysfunction and increased hsp70 expression, which are able, independently or together, to induce apoptosis. Thus, we attempted to elucidate some key apoptosis-related genes (ARG) and apoptosis-related proteins (ARP) and their expression during different stages of embryonic development, as well as to characterize the chronology of ARG expression and ARP contents after UVB radiation insult. We demonstrate that p53, Bax and Caspase3 genes are active in the embryonic cells at early embryonic developmental stages, and that the Bcl2 gene is active from the mid-embryonic stage. After UVB radiation exposure, we found an increase in ARP such as p53 and Bak after 3h of exposure. Moreover, an increase in ARG transcript levels for p53, Bax, Bcl2 and Caspase3 was observed at 6h after UVB exposure. Then, after 12h of UVB radiation exposure, an increase in Caspase3 gene expression and protein was observed, concomitantly with an increased number of apoptotic cells. Our data reveal that ARG and ARP are developmentally regulated in embryonic cells of M. olfersii and that UVB radiation causes apoptosis after 12h of exposure. Overall, we demonstrate that embryonic cells of M. olfersii are able to active the cell machinery against environmental changes, such as increased incidence of UVB radiation in aquatic ecosystems.

Homocysteine-induced changes in cell proliferation and differentiation in the chick embryo spinal cord: implications for mechanisms of neural tube defects (NTD).

Maternal hyperhomocysteinemia during pregnancy is associated with increased risk of NTD in the offspring. Our study investigated the effects of homocysteine (Hcy) on proliferation and neuronal differentiation of the spinal cord cells in a chick embryo model. Embryos were treated with 20µmol D-L Hcy/50µL saline solution at embryonic day 2 (E2) and analyzed at embryonic days 4 (E4) and 6 (E6). Control embryos received exclusively 50µL saline solution. We performed immunolocalization and flow cytometry analyses using antibodies anti-phosphohistone H3 (pH3), anti-proliferating cell nuclear antigen (PCNA), anti-ß-tubulin III and anti-p53. Our results revealed that Hcy interferes in the proliferation of the neural cells, and that this effect is age-dependent and differed between Hcy-treated embryos with and without NTD. Also, Hcy induced a decrease of neuronal differentiation in the spinal cord at both embryonic ages. These findings contribute to clarifying the cellular bases of NTD genesis, under experimental hiperhomocysteinemia.

Identification and evaluation of reference genes for expression studies by RT-qPCR during embryonic development of the emerging model organism, Macrobrachium olfersii.

RT-qPCR is a sensitive and highly efficient technique that is widely used in gene expression analysis and to provide insight into the molecular mechanisms underlying embryonic development. The freshwater prawn, Macrobrachium olfersii is an emerging model organism, but, the stable reference genes of this species need to be identified and validated for RT-qPCR analysis. Thus, the aim of this study was to evaluate the expression stability of six genes (ß-act, GAPDH, EF-1α, RpL8, RpS6, AK) in embryos and in adult tissues (cerebral ganglia, muscle and hepatopancreas) of M. olfersii. The expression stabilities of these genes were evaluated using geNorm, NormFinder, BestKeeper, ΔCt method and integrated tool RefFinder. In the general ranking, RpL8 and RpS6 were the most stable genes in embryos, while RpS6 and RpL8 were the most stable in a combined adult tissue analysis. Analysis of the adult tissues revealed that ß-act and AK were the most stable genes in cerebral ganglia, RpL8 and AK in muscle, and RpS6 and ß-act in hepatopancreas. EF-1α and GAPDH were the least stable genes and as normalizer genes in RT-qPCR affected expression of the Distal-less gene during M. olfersii development. This study provides suitable reference genes for RT-qPCR analysis and allows future studies of the gene expression in M. olfersii for understanding the molecular mechanisms of their development. To our knowledge, this is the first published study that identifies and evaluates reference genes for RT-qPCR analysis in M. olfersii and could be useful as basis for evaluations of reference genes in other prawns.

Exploring developmental gene toolkit and associated pathways in a potential new model crustacean using transcriptomic analysis.

The crustaceans are one of the largest, most diverse, and most successful groups of invertebrates. The diversity among the crustaceans is also reflected in embryonic development models. However, the molecular genetics that regulates embryonic development is not known in those crustaceans that have a short germ-band development with superficial cleavage, such as Macrobrachium olfersi. This species is a freshwater decapod and has great potential to become a model for developmental biology, as well as for evolutionary and environmental studies. To obtain sequence data of M. olfersi from an embryonic developmental perspective, we performed de novo assembly and annotation of the embryonic transcriptome. Using a pooling strategy of total RNA, paired-end Illumina sequencing, and assembly with multiple k-mers, a total of 25,636,097 pair reads were generated. In total, 99,751 unigenes were identified, and 20,893 of these returned a Blastx hit. KEGG pathway analysis mapped a total of 6866 unigenes related to 129 metabolic pathways. In general, 21,845 unigenes were assigned to gene ontology (GO) categories: molecular function (19,604), cellular components (10,254), and biological processes (13,841). Of these, 2142 unigenes were assigned to the developmental process category. More specifically, a total of 35 homologs of embryonic development toolkit genes were identified, which included maternal effect (one gene), gap (six), pair-rule (six), segment polarity (seven), Hox (four), Wnt (eight), and dorsoventral patterning genes (three). In addition, genes of developmental pathways were found, including TGF-ß, Wnt, Notch, MAPK, Hedgehog, Jak-STAT, VEGF, and ecdysteroid-inducible nuclear receptors. RT-PCR analysis of eight genes related to embryonic development from gastrulation to late morphogenesis/organogenesis confirmed the applicability of the transcriptome analysis.

Developmental effects of exposure to ultraviolet B radiation on the freshwater prawn Macrobrachium olfersi: Mitochondria as a target of environmental UVB radiation.

In South America, increased UVB radiation has become an important environmental issue that is potentially threatening aquatic ecosystems. Considering that species exhibit different degrees of sensitivity to UVB radiation and that embryos are more sensitive than organisms at later life stages, the aim of this study was to characterize the effects of UVB radiation on subcellular compartments of embryos of the freshwater prawn Macrobrachium olfersi. This species lives and reproduces in clear and shallow waters, where UV radiation can fully penetrates. Embryos were irradiated with a UVB 6W lamp for 30min and examined after 1h, 12h, 24h and 48h of exposure. The irradiance of the UVB used simulates the UV radiation that embryos receive in the natural environment. The subcellular compartment most affected by the UVB radiation was the mitochondria, which exhibited a circular shape, a decrease in mitochondrial cristae, rupture of membranes and a morphology compatible with fission. These impairments were observed simultaneously with increased ROS production, just after 1h of UVB exposure. Thus, we investigated proteins related to mitochondrial fission (Drp-1) and fusion (Mfn-1), which are essential to cell maintenance. We found a significant increase in Drp-1 expression at all analyzed time-points and a significant decrease in Mfn-1 expression only after 24h of UVB exposure. Additionally, a decrease in embryonic cell viability was verified via the mitochondrial integrity assay. To conclude, we observed important mitochondrial dysfunctions against the environmental stress caused by UVB radiation. Moreover, the cellular responses found are critical and should not be disregarded, because they impact embryos that can potentially compromise the aquatic ecosystems.

Light-mediated DNA Repair Prevents UVB-induced Cell Cycle Arrest in Embryos of the Crustacean Macrobrachium olfersi.

High levels of ultraviolet-B (UVB) radiation can negatively affect aquatic animals. Macrobrachium olfersi is a prawn that lives in clear freshwaters and during the breeding season, females carry eggs in an external brood pouch. Therefore, we hypothesize that eggs are also exposed to environmental UVB radiation. The aim of this study was to investigate whether UVB radiation induces DNA damage and compromises cell cycle in embryos of M. olfersi. In laboratory, UVB irradiance (310 mW. cm(-2) ) that embryos receive in the natural environment was simulated. After irradiation, embryos were kept under different light conditions in order to recognize the presence of cell repair. UVB radiation induces DNA damage, specifically thymine dimers. After 48 h of UVB exposure, a significant decrease in the level of these dimers was observed in embryos kept under visible light while it remained constant in the dark. Moreover, under visible light and darkness, a decrease in proliferation was observed after 48 h of irradiation. An increase in PCNA expression and decrease in p53 expression were observed after, respectively, 1 and 48 h of exposure. Our results showed that UVB radiation disturbs the cell cycle and induces DNA damage in M. olfersi embryos. However, under visible light these embryos showed successful DNA repair.

Ontogeny of the digestive tract of Centropomus parallelus larvae.

Development of the digestive tract and accessory glands of larvae of the fat snook Centropomus parallelus was examined under light microscopy, from hatching to 60 day post-hatching (dph). At hatching, the digestive tract is straight and composed by a cubic cell layer. The exogenous feeding starts at 3 dph, concomitantly with the mouth opening and subdivision of the rudimentary stomach and esophagus. At 4 dph, the intestine has three sectins, and vacuoles are observed in the posterior section, indicating the beginning of protein digestion and absorption. The pharyngeal teeth appear at 9 dph, and goblet cells appear at 13 dph in the esophagus. Gastric glands appear at 30 dph, marking the beginning of weaning. The disappearance of supranuclear vacuoles in the posterior intestine occurs at 35 dph, suggesting efficiency of extracellular digestion. This study shows that C. parallelus larvae is able to start weaning 15 days earlier than reported in earlier studies, increasing the success of larviculture.

Effects of homocysteine on mesenchymal cell proliferation and differentiation during chondrogenesis on limb development.

High levels of homocysteine (Hcy) are related to an increased risk of the occurrence of congenital anomalies, including limb defects. However, few evaluations about how toxic levels of Hcy affect limb development have been reported. We investigated whether Hcy can affect the cell cycle proteins and proteins involved in mesenchymal cell differentiation during limb development, in a chicken embryo model. Embryos were treated with 20 µmol d-l Hcy/50 µl saline at embryonic day 2 and analyzed at embryonic day 6. Untreated control embryos received exclusively 50 µl saline solution. To identify cells in proliferation and cell cycle proteins, as well as Pax1/9 and Sox9 proteins, we performed immunolocalization and flow cytometry analyses using the antibodies anti-phosphohistone H3, anti-p53, anti-p21, anti-proliferating cell nuclear antigen, anti-Pax1, anti-Pax9 and anti-Sox9. No significant differences in cell proliferation were observed between Hcy-treated and untreated embryos. We observed a decrease of the proliferating cell nuclear antigen and p21 proteins, both involved in the G1 phase of cell cycle progression. On the other hand, in mesenchymal cells of the limbs, Hcy induces an increase of p53 protein, which can be activated by DNA damage. In cell differentiation, Hcy induced an increase mainly of Pax9 and Sox9 proteins. Our data indicate that the treatment with Hcy changes the mesenchymal cell dynamics during limb development, but does not change the morphology of the cartilage molds. These findings provide information to understand better the cellular basis of the toxicity of Hcy on chondrogenesis during limb development.

MeHg Developing Exposure Causes DNA Double-Strand Breaks and Elicits Cell Cycle Arrest in Spinal Cord Cells.

The neurotoxicity caused by methylmercury (MeHg) is well documented; however, the developmental neurotoxicity in spinal cord is still not fully understood. Here we investigated whether MeHg affects the spinal cord layers development. Chicken embryos at E3 were treated in ovo with 0.1 µg MeHg/50 µL saline solution and analyzed at E10. Thus, we performed immunostaining using anti-γ-H2A.X to recognize DNA double-strand breaks and antiphosphohistone H3, anti-p21, and anti-cyclin E to identify cells in proliferation and cell cycle proteins. Also, to identify neuronal cells, we used anti-NeuN and anti-ßIII-tubulin antibodies. After the MeHg treatment, we observed the increase on γ-H2A.X in response to DNA damage. MeHg caused a decrease in the proliferating cells and in the thickness of spinal cord layers. Moreover, we verified that MeHg induced an increase in the number of p21-positive cells but did not change the cyclin E-positive cells. A significantly high number of TUNEL-positive cells indicating DNA fragmentation were observed in MeHg-treated embryos. Regarding the neuronal differentiation, MeHg induced a decrease in NeuN expression and did not change the expression of ßIII-tubulin. These results showed that in ovo MeHg exposure alters spinal cord development by disturbing the cell proliferation and death, also interfering in early neuronal differentiation.

Neuronal nitric oxide synthase and its interaction with soluble guanylate cyclase is a key factor for the vascular dysfunction of experimental sepsis.

OBJECTIVE: Vascular dysfunction plays a central role in sepsis, and it is characterized by hypotension and hyporesponsiveness to vasoconstrictors. Nitric oxide is regarded as a central element of sepsis vascular dysfunction. The high amounts of nitric oxide produced during sepsis are mainly derived from the inducible isoform of nitric oxide synthase 2. We have previously shown that nitric oxide synthase 2 levels decrease in later stages of sepsis, whereas levels and activity of soluble guanylate cyclase increase. Therefore, we studied the putative role of other relevant nitric oxide sources, namely, the neuronal (nitric oxide synthase 1) isoform, in sepsis and its relationship with soluble guanylate cyclase. We also studied the consequences of nitric oxide synthase 1 blockade in the hyporesponsiveness to vasoconstrictors. DESIGN: Randomized controlled prospective experimental study. SETTING: Academic research laboratory. SUBJECTS: Female Wistar rats submitted to cecal ligation and puncture method. INTERVENTIONS: 1) Six, 12, and 24 hours after cecal ligation and puncture, vascular reactivity to phenylephrine (3 and 30 nmol/kg) before and after 7-nitroindazole (45 µmol/kg, s.c.) or aminoguanidine (30 µmol/kg, s.c.) administration was evaluated. 2) Protein levels and interaction between nitric oxide synthase 1 and soluble guanylate cyclase were determined. 3) Six, 12, and 24 hours after cecal ligation and puncture, thoracic aorta segments were stimulated with phenylephrine in the presence or absence of 7-nitroindazole and cyclic guanosine monophosphate accumulation was determined. 4) After 24 hours of cecal ligation and puncture, norepinephrine was infused (10 µg/kg/min) in the presence or absence of 7-nitroindazole or S-methyl-L-thiocitrulline (1 µmol/kg, IV) and mean arterial pressure was registered. MEASUREMENTS AND MAIN RESULTS: 1) Both nitric oxide synthase 1 and soluble guanylate cyclase are expressed in higher levels in vascular tissues during sepsis; 2) both proteins physically interact and nitric oxide synthase 1 blockade inhibits cyclic guanosine monophosphate production; 3) pharmacological blockade of nitric oxide synthase 1 using 7-nitroindazole or S-methyl-L-thiocitrulline reverts the hyporesponsiveness to phenylephrine and increases the vasoconstrictor effect of norepinephrine and phenylephrine. CONCLUSIONS: Sepsis induces increased expression and physical association of nitric oxide synthase 1/soluble guanylate cyclase and a higher production of cyclic guanosine monophosphate that together may help explain sepsis-induced vascular dysfunction. In addition, selective inhibition of nitric oxide synthase 1 restores the responsiveness to vasoconstrictors. Therefore, inhibition of nitric oxide synthase 1 (and possibly soluble guanylate cyclase) may represent a valuable alternative to restore the effectiveness of vasopressor agents during late sepsis.

Changes in ultrastructure and expression of steroidogenic factor-1 in ovaries of zebrafish Danio rerio exposed to glyphosate.

Glyphosate is a broad-spectrum organophosphate (OP) herbicide, highly soluble in water, and when applied in terrestrial systems it penetrates into soil, eventually reaching the aquatic community and affecting nontarget organisms. The aim of this study was to evaluate the toxicity of glyphosate on ovaries of zebrafish (Danio rerio). Ovaries (n = 18 per triplicate) were exposed to 65 µg/L of glyphosate [N-(phosphonomethyl) glycine] for 15 d. This concentration was determined according to Resolution 357/2005/CONAMA/Brazil, which establishes the permissible concentration of glyphosate in Brazilian inland waters. Nonexposed ovaries (n = 18 per triplicate) were used as control. Subsequently, morphology and expression of steroidogenic factor-1 (SF-1) of exposed and nonexposed ovaries was determined. No apparent changes were noted in general morphology of exposed and nonexposed ovaries. However, a significant increase in diameter of oocytes was observed after exposure to glyphosate. When ovarian ultrastructure was examined the presence of concentric membranes, appearing as myelin-like structures, associated with the external membranes of mitochondria and with yolk granules was found. After glyphosate exposure, immunohistochemistry and immunoblotting revealed greater expression of SF-1 in the oocytes, which suggests a relationship between oocyte growth and SF-1 expression. These subtle adverse effects of glyphosate on oocytes raised a potential concern for fish reproduction. These results contribute to understanding glyphosate-induced toxicity to nontarget organisms, showing subcellular and molecular impairments that may affect reproduction in +female fish.

On the brain of a crustacean: a morphological analysis of CaMKII expression and its relation to sensory and motor pathways.

Calcium/calmodulin kinase II (CaMKII) is a Ca(2+)-activated enzyme that is abundant in vertebrate and invertebrate brains. However, its characterization is poorly addressed in the nervous system of crustaceans, and, to our knowledge, no studies have determined the microanatomical location of CaMKII in a crustacean species. In this study, we found labeling of CaMKII in the eyestalk and brain of the prawn Macrobrachium acanthurus, by means of immunohistochemistry and Western blotting. Antibodies against neuron (ß tubulin III), glutamate receptor (GluA1), and FMRFamide were used in order to further characterize the CaMKII-labeled cells in the brain. In the eyestalk, strong labeling with CaMKII was observed in the photoreceptors. These cells, especially in the rhabdom, were also reactive to anti-ß tubulin III, whereas the pigment cells were labeled with anti-CaMKII. GluA1 co-located with CaMKII in the photoreceptors. Also, CaMKII appeared in the same sites as FMRFamide in the deutocerebrum, including the olfactory lobe, and in the tritocerebrum, specifically in the antennular neuropil, indicating that the synaptic areas in these regions may be related to sensory-motor processing. In the brain, the identification of cells and regions that express CaMKII contributes to the understanding of the processing of neural connections and the modulating role of CaMKII in decapod crustaceans.

Homocysteine causes disruptions in spinal cord morphology and changes the expression of Pax 1/9 and Sox 9 gene products in the axial mesenchyme.

BACKGROUND: Neural tube defects (NTD) involve disruptions in the axial mesenchyme, and are related to an imbalance between folic acid (FA) and homocysteine (Hcy). This study evaluated the effects of FA/Hcy imbalance on cell proliferation and expression of the Pax 1/9 and Sox 9 gene products in the axial mesenchyme of chickens. METHODS: Embryos were incubated (38°C) and pretreated at 24 h and treated at 46 h of incubation. The experimental groups were: FA-pretreated with saline and treated with 0.5 µg FA/saline; Hcy-pretreated with 50 µl saline and treated with 20 µmol D,L-Hcy/50 µl saline; FA+Hcy-pretreated with 0.5 µg FA/50 µl saline and treated with 20 µmol D,L-Hcy/50 µl saline; and the control embryos were pretreated and treated with saline. Embryos were analyzed at E4 and E6. Immunohistochemistry was performed to identify proliferating cells and the expression of the gene products of Pax 1/9 and Sox 9. Total RNA of the E4 embryos was extracted and a RT-qPCR assay was performed to quantify Pax 1/9 mRNA expression. RESULTS: Hcy treatment caused spinal NTD and abnormalities in axial mesenchyme development, affecting the distribution of sclerotomal cells and chondrification. Hcy also reduced cell proliferation and changed the expression of Pax 1/9 and Sox 9 in the mesenchyme. CONCLUSIONS: Our data clarified the relationship between spinal NTD genesis and disruptions of Pax 1/9 and Sox 9 gene products in the axial mesenchyme caused by the FA/Hcy imbalance.

Prenatal lead acetate exposure induces apoptosis and changes GFAP expression during spinal cord development.

Lead is an important heavy metal pollutant in the environment, and it induces neurodevelopmental toxicity, which is characterized by histological, ultrastructural, and neurochemical changes in the central nervous system. The aim of this study was to evaluate the effects of prenatal acute lead exposure on apoptosis, GFAP expression, and lead deposition in the developing spinal cord. Chick embryos were exposed to 150µg or 450µg doses of lead acetate via yolk sac at E3 or E5 embryonic ages and incubated for six days. Lead deposition was observed in the ependymal cells, developing dorsal, and ventral horns, and in the white matter of all the exposed embryos. TUNEL-positive cells were found in all layers of the spinal cord of the control and treated embryos, and lead exposure resulted in a significant increase in the numerical density of the apoptotic cells. Control embryos showed intense GFAP expression in the ependymal cells of the roof and floor plates, and in the gray and white matters; whereas exposure to lead reduced GFAP reactivity. In ovo lead exposure induces apoptosis, and reduces GFAP expression in the nervous system of the chick embryos, which may cause impairments during neuronal development and consequences in childhood and adulthood.