Brain Of Male Zebrafish Research Articles

Environmental levels of azoxystrobin disturb male zebrafish behavior: Possible roles of oxidative stress, cholinergic system, and dopaminergic system

A widely applied pesticide of azoxystrobin, is increasingly detected in the water environment. Concern has been raised against its potential detriment to aquatic ecosystems. It has been shown that exposure to azoxystrobin interfere with the locomotor behavior of zebrafish larvae. This study aims to investigate whether exposure to environmental levels of azoxystrobin (2 μg/L, 20 μg/L, and 200 μg/L) changes the behavior of male adult zebrafish. Herein, we evaluated behavioral response (locomotor, anxiety-like, and exploratory behaviors), histopathology, biochemical indicators, and gene expression in male adult zebrafish upon azoxystrobin exposure. The study showed that exposure to azoxystrobin for 42 days remarkably increased the locomotor ability of male zebrafish, resulted in anxiety-like behavior, and inhibited exploratory behavior. After treatment with 200 μg/L azoxystrobin, vasodilatation, and congestion were observed in male zebrafish brains. Exposure to 200 μg/L azoxystrobin notably elevated ROS level, MDA concentration, CAT activity, and AChE activity, while inhibiting SOD activity, GPx activity, ACh concentration, and DA concentration in male zebrafish brains. Moreover, the expression levels of genes related to the antioxidant, cholinergic, and dopaminergic systems were significantly changed. This suggests that azoxystrobin may interfere with the homeostasis of neurotransmitters by causing oxidative stress in male zebrafish brains, thus affecting the behavioral response of male zebrafish.

Dydrogesterone disrupts lipid metabolism in zebrafish brain: A study based on metabolomics and Fourier transform infrared spectroscopy

Brain is a potential target for neuroprogestogens and/or peripheral progestogens. Previous studies reported that expression of genes about steroidogenesis, reproduction, cell cycle, and circadian rhythm in zebrafish brain could be affected by progestogens. However, there are limited information from metabolites or biomacromolecules aspects, leaving an enormous gap in understanding toxic effects of progestogens on fish brain. In this study, we exposed zebrafish embryos to 2.8, 27.6, and 289.8 ng/L dydrogesterone (DDG, a synthetic progestogen) until sexual maturity (140 days). LC-MS and GC-MS based untargeted metabolomics and Fourier-transform infrared (FTIR) spectroscopy were then performed to investigate the metabolic profiles and macromolecular changes of brain of these zebrafish. The results from multivariate statistical analysis of metabolite features showed a clear separation between different treatment groups of both female and male zebrafish brains. DDG exposure increased the levels of cholesterol, saturated fatty acids, and nucleoside monophosphates, but decreased the contents of polyunsaturated fatty acids (PUFAs), lysophosphatides, and nucleosides in dose-dependent manner. FTIR results indicated that DDG exposure led to accumulation of saturated lipids, reduction of nucleic acids and carbohydrates, and alteration of protein secondary structures. The findings from this study demonstrated that DDG could affect contents of metabolites and biomacromolecules of zebrafish brain, which may finally lead to brain dysfunctions.

Automated Capillary Isoelectric Focusing-Tandem Mass Spectrometry for Qualitative and Quantitative Top-Down Proteomics.

Top-down proteomics (TDP) aims to delineate proteomes in a proteoform-specific manner, which is vital for accurately understanding protein function in cellular processes. It requires high-capacity separation of proteoforms before mass spectrometry (MS) and tandem MS (MS/MS). Capillary isoelectric focusing (cIEF)-MS has been recognized as a useful tool for TDP in the 1990s because cIEF is capable of high-resolution separation of proteoforms. Previous cIEF-MS studies concentrated on measuring the protein's mass without MS/MS, impeding the confident proteoform identification in complex samples and the accurate localization of post-translational modifications on proteoforms. Herein, for the first time, we present automated cIEF-MS/MS-based TDP for large-scale delineation of proteoforms in complex proteomes. Single-shot cIEF-MS/MS identified 711 proteoforms from an Escherichia coli (E. coli) proteome consuming only nanograms of proteins. Coupling two-dimensional size-exclusion chromatography (SEC)-cIEF to ESI-MS/MS enabled the identification of nearly 2000 proteoforms from the E. coli proteome. Label-free quantitative TDP of zebrafish male and female brains using SEC-cIEF-MS/MS quantified thousands of proteoforms and revealed sex-dependent proteoform profiles in brains. Particularly, we discovered several proteolytic proteoforms of pro-opiomelanocortin and prodynorphin with significantly higher abundance in male zebrafish brains as potential endogenous hormone proteoforms. Multilevel quantitative proteomics (TDP and bottom-up proteomics) of the brains revealed that the majority of proteoforms having statistically significant difference in abundance between genders showed no abundance difference at the protein group level. This work represents the first multilevel quantitative proteomics study of sexual dimorphism of the brain.

Expression profile of glucose transport-related genes under chronic and acute exposure to growth hormone in zebrafish

The brain is a highly demanding organ in terms of energy requirements, and precise regulatory mechanisms must operate to ensure adequate energy delivery to maintain normal neuronal activity. Of the energy-promoting substrates present in the circulation, glucose is preferred by the brain, and as with all other substrates, its utilization depends on the presence of humoral factors such as hormones including growth hormone (GH). Glucose enters the cells though specific transport proteins. Among all transporter families and subtypes described to date, the most studied ones are the glucose transporters (GLUTs). The aim of this study is to determine a possible relationship between GH and GLUTs. Therefore, we evaluated the effect of GH-transgenesis and recombinant GH injections upon GLUT expression in the brain of male zebrafish. Overall, the results demonstrated that increasing the GH concentrations above the normal level, via transgenesis or injection, in the fish may impair energy uptake by the brain. This appeared to occur through downregulation of most of the analyzed GLUTs.

An embryonic 100 μg/L lead exposure results in sex-specific expression changes in genes associated with the neurological system in female or cancer in male adult zebrafish brains

Developmental lead (Pb) exposure is linked to neurological health issues. Results from non-human primate and rodent studies suggest detrimental effects of an early life Pb exposure, showing transcriptional disturbances and pathological evidence of Alzheimer's disease in the adult animal brain. To elucidate the impacts of an embryonic Pb exposure on the adult brain, transcriptomic analysis was completed on the brain of zebrafish aged 12months exposed to a control treatment or to an embryonic 100μg/L Pb exposure by sex. In the adult female zebrafish brain, significant changes in expression profiles occurred in a number of genes involved in neurological disease and nervous system development and function. On the other hand, in adult males, a number of genes with significant expression alterations were found to be associated with cancer and tumors. p38 mitogen-activated protein kinase (p38 MAPK) was also indicated as an upstream regulator of observed gene expression changes. Western blot analysis confirmed activation of p38 MAPK in the form of phosphorylated p38 MAPK in the male zebrafish brain. In addition, we compared transcriptomic changes observed in this study to a previous study with an embryonic exposure of 10μg/L Pb by sex, showing unique sets of genes dependent on Pb concentration. Overall, these results show sex-specific and concentration-dependent disturbances of global gene expression patterns in the brain of adult zebrafish exposed to Pb during embryogenesis.

Proteomics analysis of zebrafish brain following chronically exposed to bisphenol A

ABSTRACTBisphenol A, a plastic monomer and plasticizer, is a well-known endocrine disrupter, widely present in the aquatic environment, but little is known regarding its neurotoxicity in fish. Herein, we investigated its effects on male zebrafish brain. Zebrafish were exposed to 10 µg/L BPA for 45 days. An isobaric tags for relative and absolute quantitation approach coupled with nano high-performance liquid chromatography-tandem mass spectrometry analysis was employed to detect and identify differentially expressed proteins. A total of 46 proteins was identified and categorized into functional classes that mostly included metabolism and transport, cytoplasm and organelle, ion and nucleotide binding, indicating that bisphenol A toxicity in fish brain is complex. The biological pathways of starch and sucrose metabolism, calcium signaling, and aminoacyl-tRNA biosynthesis were also induced. Proteomic analyses add new perspectives to bisphenol A neurotoxicity in aquatic organisms.

Temperature differentially regulates the two kisspeptin systems in the brain of zebrafish

Kisspeptins encoded by the kiss1 and kiss2 genes play an important role in reproduction through the stimulation of gonadotropin-releasing hormone (GnRH) secretion by activating their receptors (KissR1 EU047918 and KissR2 EU047917). To understand the mechanism through which temperature affects reproduction, we examined kiss1 and kiss2 and their respective receptor (kissr1 and kissr2) gene expression in the brain of male zebrafish exposed to a low temperature (15°C), normal temperature (27°C), and high temperature (35°C) for 7-days. kiss1 mRNA levels in the brain were significantly increased (2.9-fold) in the low temperature compared to the control (27°C), while no noticeable change was observed in the high temperature conditions. Similarly, kissr1 mRNA levels were significantly increased (1.5–2.2-folds) in the low temperature conditions in the habenula, the nucleus of the medial longitudinal fascicle, oculomotor nucleus, and the interpeduncular nucleus. kiss2 mRNA levels were significantly decreased (0.5-fold) in the low and high temperature conditions, concomitant with kissr2 mRNA levels (0.5-fold) in the caudal zone of the periventricular hypothalamus and the posterior tuberal nucleus. gnrh3 but not gnrh2 mRNA levels were also decreased (0.5-fold) in the low and high temperature conditions. These findings suggest that while the kiss1/kissr1 system is sensitive to low temperature, the kiss2/kissr2 system is sensitive to both extremes of temperature, which leads to failure in reproduction.

Proteomic analysis of zebrafish brain tissue following exposure to the pesticide prochloraz

The hypothalamus–pituitary–gonadal (HPG) axis plays a central role in the maintenance of homeostasis and disruptions in its function can have important implications for reproduction and other critical biological processes. A number of compounds found in aquatic environments are known to affect the HPG axis. In the present study, we used two-dimensional electrophoresis to investigate the proteome of female and male zebrafish brain after 96 h exposure to the fungicide prochloraz. Prochloraz has known effects on a number of key HPG molecules, including antagonism of Cyp17 and Cyp19 (aromatase). Twenty-eight proteins were shown to be differentially expressed in the brains of females and 22 in males. Proteins were identified using LC–MS/MS and identities were examined relative to brain function in the context of changing steroid hormone levels. There was little overlap between sexes in proteins exhibiting differential expression. Proteins with known roles in metabolism, learning, neuroprotection, and calcium regulation were determined to be differentially regulated. Relationships between identified proteins were also examined using Ingenuity Pathway Analysis, and females were shown to exhibit enrichment of several metabolic pathways. We used differentially expressed proteins to establish a putative classifier consisting of three proteins that was able to discriminate prochloraz-exposed from control females. Putatively impacted brain functions and specific protein changes that were observed have the potential to be generalized to other that similarly impact steroid hormone levels.