Comprehensive analysis of differences of N6-methyladenosine of lncRNAs between atrazine-induced and normal Xenopus laevis testis.

BACKGROUND: Increasing evidence suggested N6-methyladenosine (m6A) modification is crucial for male germline development. However, m6A modification of lncRNAs gains a little attention in amphibians in recent years. Xenopus laevis (X. laevis) was chosen to be an ideal model organism for testing environmental endocrine disrupting chemicals (EDCs) exposure and resultant effects. Atrazine (AZ) as an endocrine disrupt can effect development of testis in amphibians. Our previous study revealed that m6A is a highly conserved modification across the species. RESULTS: The results of m6A sequences showed that m6A-methylated lncRNAs enriched in intergenic region in testes of X. laevis. We further examined the differential expression of lncRNAs m6A sites in testes of AZ-exposed and compared with that in animals from control group. The results indicated that up to 198 differentially methylated m6A sites were detected within 188 lncRNAs, in which 89 significantly up-methylated sites and 109 significantly down-methylated sites. Data from KEGG pathway analysis indicated that AZ-affected lncRNAs m6A sites were mainly involved in 10 pathways in which 3 mutual pathways were found in the result of differentially m6A-methylated mRNAs. CONCLUSIONS: These findings suggested that differentially m6A-methylated lncRNAs and these 3 pathways may act on regulatory roles in abnormal testis development of AZ-exposed X. laevis. This study for the first time provides insights into the profile of lncRNAs m6A modifications in amphibian species.

Distinct m6A methylome profiles in poly(A) RNA from Xenopus laevis testis and that treated with atrazine.

Recent discovery of reversible N6-methyladenosine (m6A) methylation on messenger RNA (mRNA) and mapping of m6A methylomes in mammals, plant and yeast revealed potential regulatory functions of this RNA modification. However, the role of the m6A methylomes in amphibious is still poorly understood. Here, we examined the m6A transcriptome-wide profile in testis tissues of Xenopus laevis (X. laevis) with and without treatment with 100 µg/L atrazine (AZ) through m6A sequencing analysis using the latest Illumina HiSeq sequencer. The results revealed that m6A is a highly conserved modification of mRNA in X. laevis. Distinct from that in mammals, m6A in X. laevisis enriched around the stop codon and start codon, as is reported in plant. We then investigated the differential expression m6A in testes of AZ-exposed X. laevis and compared that with the X. laevis in the control group by m6A sequencing. The results indicated that AZ leads to altered expression profile in 1380 m6A modification sites (696 upregulated and 684 downregulated). KEGG pathway analysis indicates that the "NOD-like receptors", "tight junction", "Peroxisome proliferator-activated receptors", "adherens junctions", "Glycerophospholipid metabolism" and "Fatty acid biosynthesis" signaling pathways may be associated with abnormal testis development of X. laevis due to exposure to AZ. Analysis results showed a positive correlation between m6A modification and mRNA abundance, suggesting a regulatory role of m6A in amphibious gene expression. Our first report of m6A transcriptome-wide map of an amphibian species X. laevis presented here provides a starting roadmap for uncovering m6A functions that may affect/control amphibian testis development.

Analysis of long non-coding RNA involved in atrazine-induced testicular degeneration of Xenopus laevis.

Long non-coding RNA (lncRNA) plays a critical role in male germline development. Atrazine (AZ) as an environmental endocrine disrupting chemical (EDCs) can induce male reproductive toxicity in amphibians. Our previous studies demonstrated that AZ can alter gene and circular RNA (circRNA) expression of damaged testes in Xenopus laevis (X. laevis). We furthered to investigate the lncRNA expression profiling in the testis of X. laevis. Over 3559 lncRNAs were detected by lncRNA sequencing. AZ induced 40 upregulated and 46 downregulated differentially expressed lncRNAs. KEGG analysis showed that AZ-affected lncRNAs mainly involve in 19 pathways among which 12 pathways are found in circRNA analysis. This study for the first time demonstrated that AZ can alter lncRNAs which may play a role in testicular degeneration through regulating expressions of functional genes in X. laevis. Our data may provide more insights on the mechanism about male reproductive toxicity of EDCs.

Validation and bioinformatics analysis of differentially expressed circRNAs involved in developing male Xenopus laevis chronically exposed to atrazine.

The data presented here are related to the research article titled "Identification of circular RNAs and their alterations involved in developing male Xenopus laevis chronically exposed to atrazine" (Sai et al., 2018) [1]. Circular RNAs (circRNAs) are implicated in multiple developmental anomalies (Bachmayr-Heyda et al., 2015; Li et al., 2015) [2], [3]. This report describes the differentially expressed circRNAs involved in developing male Xenopus laevis (X. laevis) chronically exposed to atrazine (AZ) database. The database contains the validation of differentially expressed circRNAs, KEGG analysis of differentially expressed circRNA-associated target genes and prediction of miRNA binding sites. These data may help to further evaluate the role of circRNAs in male X. laevis chronically exposed to AZ.

Identification of circular RNAs and their alterations involved in developing male Xenopus laevis chronically exposed to atrazine.

Atrazine (AZ) is an environmental endocrine disrupting chemical which can affect the development of amphibians. In our past studies, we demonstrated that chronical exposure to 100 µg/L AZ can cause abnormalities in development and related genes expression of gonads in developing male Xenopus laevis (X. laevis) tadpoles. Recent studies by others have demonstrated that circular RNAs (circRNAs) are implicated in multiple developmental anomalies. However, whether circRNAs involve in the effects in AZ-exposed X. laevis remains unknown. In this study, over 68575 circRNAs were detected by circRNA sequencing of testis tissues from control groups (n = 3) and AZ-treated X. laevis (n = 3). Treatment of AZ led to 405 circRNAs differentially expressed including 44 upregulated and 361 downregulated compared with froglets in the control groups. Two upregulated and 6 downregulated circRNAs were further validated by real-time PCR assay which displayed consistent regulation patterns as shown by the transcriptome sequencing results. Two hundreds and eighty two differentially expressed circRNAs played miRNA sponges roles. Kyoto Encyclopedia of Genes and Genomes pathway analysis of miRNA targets showed that AZ-affected circRNAs are mainly involved in 19 pathways. The Wnt signaling pathway and progesterone-mediated oocyte maturation pathway may be two involved signal pathways. This study for the first time provides evidence that AZ can alter circRNAs which play a role in AZ-induced testicular degeneration of developing male X. laevis through regulation of expressions of functional genes in the testes of X. laevis.

Continued Studies on the Effects of Simazine on the Liver Histological Structure and Metamorphosis in the Developing Xenopus laevis.

This study continued our previous work (Sai et al. in Bull Environ Contam Toxicol 95:157-163, 2015a) by analysing the effects of simazine on the liver histological structure and metamorphosis in the developing Xenopus laevis. Tadpoles (Nieuwkoop-Faber stage 46) were exposed to simazine at 0.1, 1.2, 11.0 and 100.9 µg/L for 100 days. When tadpoles were exposed to simazine at 11.0 and 100.9 µg/L, an increased mortality and damaged liver tissues were observed together with significant inhibition of percent of X. laevis completing metamorphosis on days 80 and 90 and prolonged time of completing metamorphosis. On the other hand, we found that simazine has no significant effects on liver weight and altered hepatosomatic index. Results of this study may be considered to inform risk assessment of the effects of simazine on the development of X. laevis.

Gene expression profiles in testis of developing male Xenopus laevis damaged by chronic exposure of atrazine.

As a widely used herbicide, atrazine (AZ) has been extensively studied for its adverse effects on the reproductive system, especially feminization in male animals. However, the relationship of gene expression changes and associated toxicological endpoints remains unclear. In this study, developing Xenopus laevis tadpoles were exposed to concentration of AZ at 0.1, 1, 10 or 100 µg/L continuously. Compared with froglets in the control group, there were no significant differences in body length, body weight, liver weight and hepatosomatic index (HSI) of males in groups treated with AZ for 90 d. At 100 µg/L AZ treatment caused a significant reduction of gonad weight and gonadosomatic index (GSI) of males (p < 0.01). In addition, AZ at all dose levels caused testicular degeneration, especially in froglets from the groups with 0.1 and 100 µg/L which exhibited U-shaped dose-response trend. We further investigated the gene expression changes associated with the testicular degeneration induced by AZ. We found that the expression of 1165 genes was significantly altered with 616 upregulated and 549 downregulated compared to the expression profile of the control animals. KEGG analysis showed that genes which were significantly affected by AZ are mainly involved in arginine and proline metabolism, cell cycle, riboflavin metabolism, spliceosome, base excision repair and progesterone-mediated oocyte maturation pathway. Our results show that AZ may affect reproductive and immune systems by interference with the related gene expression changes during the male X. laevis development. The findings may help to clarify the feminization mechanisms of AZ in male X. laevis.

The Effects of Simazine, a Chlorotriazine Herbicide, on the Expression of Genes in Developing Male Xenopus laevis.

Simazine was investigated for gene expression concurrent with simazine-induced phenotype changes during development of male Xenopus laevis. X. laevis tadpoles (Nieuwkoop-Faber stage 46) were exposed to 0.1, 1.2, 11.0 and 100.9 µg/L simazine for 100 days. The results showed that an increased mortality of X. laevis, decreased gonad weight and altered gonadosomatic index of males significantly (p<0.05) when exposed to simazine at 11.0 and 100.9 µg/L. Significant degeneration in testicular tissues was observed when tadpoles were exposed to simazine at 100.9 µg/L. To investigate the molecular mechanisms behind the testicular degeneration by simazine, we evaluated gene expression in animals treated with 100.9 µg/L simazine and found that 1,315 genes were significantly altered (454 upregulated, 861 downregulated). Genes involved in the cell cycle control, and amino acid metabolism pathways were significantly downregulated. These results indicate that simazine affects the related gene expressions which may be helpful for the understanding of the reason for the reproductive toxicity of simazine on male X. laevis.

Assessing atrazine-induced toxicities in Bufo bufo gargarizans Cantor.

Atrazine (AZ), a widely used herbicide has drawn attentions for its potential impacts on amphibians. This study aims to investigate the toxicity of AZ in Bufo bufo gargarizans Cantor (B. bufo gargarizans), a species of toad commonly found in China and countries in East Asia. We treated tadpoles with 0.1, 1, 10 and 100 µg/L AZ for 85 days and examined related parameters. The results showed that the mortality of the toads in the treatment group increased dramatically in a U-shaped dose-response relationship. The hindlimb extension and metamorphosis rate of the toads were significantly inhibited by AZ at 10 and 100 µg/L. Under the same condition, there were significant progressive changes in the testicular structures. Moreover, we found that AZ has no significant effects on growth, sex ratios, gonadal morphology, forelimb emergence and histology in the ovaries. Our results support the idea that environmental contaminants including AZ may be relevant to global amphibian decline.