Early-Life Exposure to Paraquat Aggravates Sex-Specific and Progressive Abnormal Non-Motor Neurobehavior in Aged Mice.

Early-life exposure to environmental neurotoxicants is known to have lasting effects on organisms. In this study, we aim to investigate the impacts of PQ exposure during early developmental stages and adult re-challenge in aged mice on non-motor neurobehavior. Two mouse models, which were exposed once during early life stage and re-exposure at adulthood, were created to explore the long-term effects of PQ on non-motor neurobehavior. As the results showed, early-life exposure to PQ caused impairment in working memory and cognitive ability in aged male mice, but not in female mice, exhibiting a sex-specific impairment. Moreover, male mice that were re-challenged with PQ at adulthood following early-life exposure also exhibited non-motor neurobehavioral disorders. Notably, re-exposure to PQ exacerbated neurobehavioral disorders and anxiety levels compared to single exposure during different life stages. Collectively, early-life exposure to PQ can result in irreversible impairments in non-motor neurobehavior and increase susceptibility to subsequent insults in male mice, but not in female mice, suggesting greater sensitivity in male rodents to PQ-induced non-motor neurobehavioral deficits.

Paraquat-induced neurogenesis abnormalities via Drp1-mediated mitochondrial fission.

Neurogenesis is a fundamental process in the development and plasticity of the nervous system, and its regulation is tightly linked to mitochondrial dynamics. Imbalanced mitochondrial dynamics can result in oxidative stress, which has been implicated in various neurological disorders. Paraquat (PQ), a commonly used agricultural chemical known to be neurotoxic, induces oxidative stress that can lead to mitochondrial fragmentation. In this study, we investigated the effects of PQ on neurogenesis in primary murine neural progenitor cells (mNPCs) isolated from neonatal C57BL/6 mice. We treated the mNPCs with 0-40 µM PQ for 24 h and observed that PQ inhibited their proliferation, migration, and differentiation into neurons in a concentration-dependent manner. Moreover, PQ induced excessive mitochondrial fragmentation and upregulated the expression of Drp-1, p-Drp1, and Fis-1, while downregulating the expression of Mfn2 and Opa1. To confirm our findings, we used Mdivi-1, an inhibitor of mitochondrial fission, which reversed the adverse effects of PQ on neurogenesis, particularly differentiation into neurons and migration of mNPCs. Additionally, we found that Mito-TEMPO, a mitochondria-targeted antioxidant, ameliorated excessive mitochondrial fragmentation caused by PQ. Our study suggests that PQ exposure impairs neurogenesis by inducing excessive mitochondrial fission and abnormal mitochondrial fragmentation via oxidative stress. These findings identify mitochondrial fission as a potential therapeutic target for PQ-induced neurotoxicity. Further research is needed to elucidate the underlying mechanisms of mitochondrial dynamics and neurogenesis in the context of oxidative stress-induced neurological disorders.

bta-miR-183 targets EZRIN to regulate microvilli formation and improve early development of bovine embryos.

In brief: Almost total lack of sperm-borne RNAs is regarded as one of the key factors that leads to the abnormal development of somatic cell nuclear transfer embryo. This paper reveals a need for us to further explore the roles of the paternal regulatory factors on embryonic development in early embryos. Abstract: Mature sperm contain both coding and non-coding RNAs, which can be delivered into an oocyte with the sperm at fertilization. Accumulating evidences show that these sperm-borne RNAs play crucial roles in epigenetic reprogramming, cytoskeleton remodeling, embryonic development, and offspring phenotype. Almost total lack of sperm-borne RNAs is regarded as one of the key factors that leads to the abnormal development of somatic cell nuclear transfer (SCNT) embryo. bta-miR-183 was found to be highly expressed in bovine sperm and can be delivered into oocytes during fertilization in our previous study, and in this study, EZR was confirmed as a target gene of bta-miR-183 in early embryos by bioinformatics, luciferase, and gain-of-function and loss-of-function experiments. Scanning electron microscopy showed that the density of microvilli on the surface of SCNT embryos was significantly higher than that onin vitro fertilized embryos and was significantly reduced by injection of bta-miR-183 mimic. EZR-siRNA injected into SCNT embryos had a similar effect. This indicated that the lack of bta-miR-183 might lead to abnormal changes in microvilli by downregulating ezrin protein. In addition, gain-of-function studies showed that bta-miR-183 significantly improved developmental competence of SCNT embryo in terms of cleavage (76.63% vs 64.32%, P < 0.05), blastocyst formation (43.75% vs 28.26%, P < 0.05), apoptotic index (5.21% vs 12.64%, P < 0.05), and the trophoblast ratio (32.65% vs 25.58%, P < 0.05) in day 7 blastocysts. Thus, the present study indicated that bta-miR-183 might play crucial roles in the formation of microvilli and embryo development by regulating expression of EZR mRNA.

Impacts of early-life paraquat exposure on gut microbiota and body weight in adult mice.

Environmental chemicals can affect the composition and metabolic functions of gut microbiota, leading to various diseases including obesity. The composition of gut microbiota is highly dynamic in the early stages of life. Increasing lines of evidence suggest the adverse effect of early onset chemical exposure on gut microbiota and adulthood body weight gain. Paraquat (PQ) is a widely used toxic herbicide. However, it remains unclear whether PQ can affect the gut microbiota, particularly exposed during early life stage and its link to obesity in adulthood. Here, we applied 16S rRNA gene sequencing to explore how the gut microbiota of adult mice changed after postnatal PQ exposure via intraperitoneal injection. In addition, the body weight of mice was monitored through adulthood. Our results showed that early-life PQ exposure increased the body weight and perturbed the gut microbiota of adult mice in a highly sex-specific manner. In males, early PQ exposure reduced gut microbiota diversity and altered the structure of gut microbiota in adulthood. Interestingly, these changes were not observed in females. Moreover, gene function prediction analysis implied that PQ-induced alteration of gut microbiota was highly correlated with body weight gain in male mice. Taken together, these results suggest that early-life PQ exposure can perturb the gut microbiota and result in increased body weight in adult male mice, which highlights the potential role of gut microbiota in the toxicity of early-life PQ exposure and its sex-specific effects.

Cadmium inhibits neural stem/progenitor cells proliferation via MitoROS-dependent AKT/GSK-3ß/ß-catenin signaling pathway.

Cadmium (Cd) is a toxic heavy metal widely found in the environment. Cd is also a potential neurotoxicant, and its exposure is associated with impairment of cognitive function. However, the underlying mechanisms by which Cd induces neurotoxicity are unclear. In this study, we investigated the in vitro effect of Cd on primary murine neural stem/progenitor cells (mNS/PCs) isolated from the subventricular zone. Our results show that Cd exposure leads to mNS/PCs G1/S arrest, promotes cell apoptosis, and inhibits cell proliferation. In addition, Cd increases intracellular and mitochondrial reactive oxygen species (ROS) that activates mitochondrial oxidative stress, decreases ATP production, and increases mitochondrial proton leak and glycolysis rate in a dose-dependent manner. Furthermore, Cd exposure decreases phosphorylation of protein kinase B (AKT) and glycogen synthase kinase-3 beta (GSK3ß) in mNS/PCs. In addition, pretreatment mNS/PCs with MitoTEMPO, a mitochondrial-targeted antioxidant, improves mitochondrial morphology and functions and attenuates Cd-induced inhibition of mNS/PCs proliferation. It also effectively reverses Cd-induced changes of phosphorylation of AKT and the expression of ß-catenin and its downstream genes. Taken together, our data suggested that AKT/GSK3ß/ß-catenin signaling pathway is involved in Cd-induced mNS/PCs proliferation inhibition via MitoROS-dependent pattern.

Single-cell RNA sequencing of mouse neural stem cell differentiation reveals adverse effects of cadmium on neurogenesis.

Cadmium (Cd) is a toxic heavy metal and widely exists in the environment. Extensive studies have revealed that Cd exposure can elicit neurotoxicity and potentially interfere with neurogenesis. However, underlying mechanisms by which Cd exposure affects neurogenesis remain unclear. In this study, we performed single-cell RNA sequencing (scRNA-seq) of the differentiated mixture from neonatal mouse Neural Stem Cells (mNSCs) that were exposed to Cd for 24 h and differentiated for 7 days. Our results showed that Cd exposure led to an increase in the differentiation of NSCs into astrocytes while a decrease into neurons. Besides, Cd induced subtype-specific response and dysregulated cell-to-cell communication. Collectively, our scRNA-seq data suggested that Cd had toxic effects on NSCs differentiation at the single-cell level, which offered insight into the potential molecular mechanism of Cd on neurogenesis. Furthermore, our findings provided a new method for assessing the neurodevelopmental toxicity of environmental pollutants.

The effects of glycine-glutamine dipeptide replaced l-glutamine on bovine parthenogenetic and IVF embryo development.

Relative to alanine and serine amino acid levels, glutamine is highly abundant in follicular fluid, and is an important source of energy required for oocyte maturation and embryo development. Thus, glutamine is an essential component of in vitro embryo culture media. However, glutamine has poor stability and degrades spontaneously in solution to form ammonia and pyrrolidonecarboxylic acid. In the present study, we aimed to explore the effect of substituting l-glutamine with glycine-glutamine, a more stable glutamine, on development of early parthenogenetic embryos and in vitro fertilization (IVF) embryos in bovine. Results revealed that glycine-glutamine can significantly increase cleavage rate (parthenogenetic embryos:87.24% vs. 72.61%, IVF embryos:89.33% vs. 83.79%, P < 0.01), blastocyst number (parthenogenetic embryos:24.98% vs. 18.07%, IVF embryos:33.53% vs. 27.29%, P < 0.01), and blastocyst number (parthenogenetic embryos:96 vs. 76, IVF embryos:114 vs. 109, P < 0.01), reduce blastocyst apoptosis (parthenogenetic embryos:3.72% vs. 6.65%, IVF embryos:2.53% vs.6.23%, P < 0.01), alleviate embryo ammonia toxicity, and reduce the content of reactive oxygen species (ROS) compared with the l-glutamine. In addition, glycine-glutamine can alter epigenetic reprogramming by increasing the expression of HDAC1 (Histone Deacetylase 1) and decreasing the relative expression levels of H3K9 acetylation in early parthenogenetic embryos and IVF embryos. From our present study, we concluded that glycine-glutamine is an effective substitute of glutamine in modified synthetic oviduct fluid with amino acids (mSOFaa).

Sperm-borne small RNAs regulate α-tubulin acetylation and epigenetic modification of early bovine somatic cell nuclear transfer embryos.

Accumulated evidence indicates that sperm-borne small RNA plays a crucial role in embryonic development, especially the absence of the sperm-borne small RNA might be a major cause of the abnormal development of cloned embryos. In this study, we found that sperm-borne small RNA can affect abnormal pronuclear-like structures, postpone the timing of first embryo cleavage and enhance developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos. In addition, the supplementation of sperm-borne small RNA can significantly increase live birth rates and decrease the birth weights of cloned offspring. To investigate the underlying mechanisms, the levels of α-tubulin K40 acetylation (Ac α-tubulin K40) and histone H3 lysine 9 trimethylation (H3K9me3) during early embryo development were investigated in SCNT embryos with sperm-borne small RNA supplementation (termed as T-NT), compared to those normal SCNT embryos and embryos obtained from standard IVF. The results showed that sperm-borne small RNA can significantly decrease the H3K9me3 levels at the pronuclear and two-cell stages, while significantly increase Ac α-tubulin K40 levels at anaphase and telophase of bovine SCNT embryos during the first cleavage. Collectively, our study for the first time demonstrates that sperm-borne small RNA plays a crucial role in the developmental competence of SCNT embryos by regulating H3K9me3 and Ac α-tubulin K40. Further studies will be required to determine how sperm small RNA regulate the H3K9me3 and Acα-tubulin K40. Our study suggests that the supplementation of sperm-borne small RNA is a potential application to improve the cloning efficiency.

Comparison of RNA extraction and microRNA detection protocols for a small amount of germinal vesicle oocytes in bovine.

RT-qPCR is a widely used method to detect miRNA expression. Compared with mRNA, miRNA has a shorter length and lower abundance which hinders the acquisition of reliable results. Thus, miRNA detection requires a method with high sensitivity and accuracy. Collecting large amounts of material is particularly difficult for oocytes and pre-implantation embryos of domestic animals. Establishing a set of miRNA detection methods that are suitable to detect trace amounts of such materials is urgently needed. In this study, the total RNA in 50 germinal vesicle (GV) oocytes was isolated through direct lysis and by using mirVana miRNA Isolation Kit and miRNeasy Micro Kit. The OD260/280 values and concentrations of the RNA in these three groups were compared to identify a superior RNA isolation method. In addition, the specificity and sensitivity of common DNA and LNA primers were compared by real-time quantitative polymerase chain reaction for miRNA detection. Results show that the RNA concentration of in the direct lysis group was significantly higher than that in the other two groups. The specificity between the DNA primers and LNA primers was identical, whereas the sensitivity of LNA primers was superior to that of DNA primers. These results suggest that direct lysis combined with LNA primers might be a suitable protocol for the miRNA detection of a small amounts of GV oocytes and pre-implantation embryos in cattle.

Bovine uterus-derived exosomes improve developmental competence of somatic cell nuclear transfer embryos.

Exosomes widely exist in various tissues and body fluids, including blood, tissue fluid, and urine. In the present study, exosomes were first isolated from the early luteal phase uterus and confirmed through morphological examination, immunofluorescence (IF) staining of special membrane antigen, and Western blot. The effects of exosomes on the developmental competence of somatic cell nuclear transfer (SCNT) embryos were investigated. Transmission electron microscopy results showed that the isolated exsomes were spherical particles with a 50 nm-150 nm diameter. Immunostaining showed that the surface of these isolated particles were CD9 positive, which was confirmed using Western blot. Supplementing SCNT embryos with these isolated exsomes on day 4 of culture significantly increased the blastocyst formation rate (31% vs. 34%, 40.3%, and 34.3%) and hatching rate (30.3% vs. 33.3%, 40.7%, and 35%) in comparison with the non-supplementation (control), and day 3 and day 5 supplementation groups. Blastocysts from the exsome supplementation groups showed higher inner cell mass/trophectoderm cell ratio (48% vs 37.9%) and lower apoptosis index (2.1% vs 6.5%) than the control group. The gene expression analysis of the blastocysts also showed that the exsomes supplementation significantly enhanced the expression levels of IFNT and acrogranin and decreased the expression levels of HSP70, BAX and BIP. In conclusion, the present study indicated that the early luteal phase uterus secretes exosomes, which might play important roles in the development of SCNT embryos.