Molecular characterization and mRNA expression of ribosomal protein L8 in Rana nigromaculata during development and under exposure to hormones

Lou, Qin-Qin; Cao, Shan; Xu, Wei; Zhang, Yinfeng; Qin, Zhan-Fen; Wei, Wuji

doi:10.1016/j.jes.2014.09.018

Cited by 16 publications

(9 citation statements)

References 37 publications

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“…The specific primers of these genes were designed using Primer Premier 6.0, according to the sequences of P. nigromaculatus that we submitted to GenBank. Ribosomal protein L8 (rpl8), the most used reference gene in amphibians, was used as a reference gene to normalize messenger RNA expression of sex‐related genes according to our previous study . In the present study, the cycle threshold ( C t ) values of rpl8 varied in a small range, with no significant differences among all treatment groups, demonstrating that rpl8 is suitable as a reference gene (Supplemental Data, Figure S2).…”

Section: Methodsmentioning

confidence: 53%

Low concentrations of dihydrotestosterone induce female‐to‐male sex reversal in the frog Pelophylax nigromaculatus

Lou

et al. 2015

Enviro Toxic and Chemistry

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Previous studies have demonstrated that some amphibian species can be sex-reversed by high concentrations of androgens. Little attention has focused on the effects of androgenic endocrine-disrupting chemicals (EDCs) on amphibians. The present study aimed to investigate the effects of lower concentrations of the androgenic EDC 5α-dihydrotestosterone (DHT) on gonadal differentiation and development in Pelophylax nigromaculatus, a true frog distributed widely in East Asia. Tadpoles at Gosner stage 24/25 were exposed to nominal concentrations of 40 ng/L, 400 ng/L, and 4000 ng/L DHT to complete metamorphosis. In all DHT treatment groups, males and ambiguous sexes were identified based on gonadal morphology, whereas no females were found; thus, all treatment groups exhibited male-skewed ratios compared with the control group. Gonadal histological examination revealed that ambiguous sexes displayed overall testicular structure with certain ovarian characteristics, demonstrating that DHT-induced sex-ambiguous gonads were incomplete ovary-to-testis reversals (IOTTRs). The expression levels of some ovary-biased genes in the IOTTRs were significantly higher than in the control testes but lower than in the control ovaries. These results show that low concentrations of DHT induced complete or incomplete female-to-male sex reversal in P. nigromaculatus, and incomplete sex reversal retained certain ovarian characteristics not only at gonadal morphological and histological levels but also at the molecular level. They present study highlights potential risks of DHT and other androgenic EDCs for P. nigromaculatus.

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Section: Methodsmentioning

confidence: 53%

Low concentrations of dihydrotestosterone induce female‐to‐male sex reversal in the frog Pelophylax nigromaculatus

Lou

et al. 2015

Enviro Toxic and Chemistry

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“…2.1. Animals and housing conditions P. nigromaculatus was raised as described in our previous studies (Li et al, 2015;Lou et al, 2014). Briefly, adult frogs were raised in amphibious aquarium containing sand and dechlorinated water, and fed with Terzebrio molitor daily.…”

Section: Methodsmentioning

confidence: 99%

“…The quantification of gene expression was based on a comparative cycle threshold (Ct) value. According to our previous study (Lou et al, 2014), ribosomal protein L8 (rpl8) was used as a reference gene to normalize mRNA expression in P. nigromaculatus. The relative transcriptional abundance of each gene was analyzed by the 2 −△△Ct method (Livak and Schmittgen, 2001).…”

Section: Rna Extraction and Gene Expression Analysismentioning

confidence: 99%

Tetrabromoethylcyclohexane affects gonadal differentiation and development in the frog Pelophylax nigromaculatus

Liu

Meng

et al. 2017

Aquatic Toxicology

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Tetrabromoethylcyclohexane (TBECH), an additive brominated flame retardant, has been shown to have an androgenic activity in vitro. In the present study, we aimed to investigate the effects of TBECH on gonadal differentiation and development in the frog Pelophylax nigromaculatus, an amphibian species sensitive to androgenic chemicals, and to assess the androgenic activity of TBECH in vivo. P. nigromaculatus tadpoles were exposed to TBECH (1, 10, 100nM) from Gosner stage 24 to complete metamorphosis, and to 5α-dihydrotestosterone (DHT) as a positive control. We found that 1nM DHT resulted in 100% males, while the sex ratio in the solvent control group was close to 1:1. In all the TBECH treatment groups, sexually ambiguous gonads based on gross morphology and intersexualities with testicular and ovarian histological structures were found, but no abnormality occurred in the solvent control. In the 1, 10, 100nM TBECH treatment groups, the female percentages were 52%, 31%, 17%, with 36%, 56%, 66% for males and 12%, 13%, 17% for abnormal sexes, respectively. X-test revealed significant differences in sex ratios between the three TBECH groups and the solvent control group, and the sex ratios in the two higher concentration groups were male-biased. These observations show that TBECH has a masculinizing effect on gonadal differentiation and development in P. nigromaculatus, suggesting an androgenic activity of TBECH in vivo. To our knowledge, this is the first study demonstrating that TBECH could induce gonadal masculinization in an animal, which raises new concerns for reproductive risk of TBECH exposure.

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“…Ribosomal protein L8 (rpl8) was used as a reference gene to normalize mRNA expression (Lou et al, 2014b). Specific primers are shown in Table 1.…”

Section: Rna Extraction and Rt-qpcrmentioning

confidence: 99%

A screening assay for thyroid hormone signaling disruption based on thyroid hormone-response gene expression analysis in the frog Pelophylax nigromaculatus

Zhang

Qin

et al. 2015

Journal of Environmental Sciences

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Amphibian metamorphosis provides a wonderful model to study the thyroid hormone (TH) signaling disrupting activity of environmental chemicals, with Xenopus laevis as the most commonly used species. This study aimed to establish a rapid and sensitive screening assay based on TH-response gene expression analysis using Pelophylax nigromaculatus, a native frog species distributed widely in East Asia, especially in China. To achieve this, five candidate TH-response genes that were sensitive to T3 induction were chosen as molecular markers, and T3 induction was determined as 0.2 nmol/L T3 exposure for 48 hr. The developed assay can detect the agonistic activity of T3 with a lowest observed effective concentration of 0.001 nmol/L and EC50 at around 0.118-1.229 nmol/L, exhibiting comparable or higher sensitivity than previously reported assays. We further validated the efficiency of the developed assay by detecting the TH signaling disrupting activity of tetrabromobisphenol A (TBBPA), a known TH signaling disruptor. In accordance with previous reports, we found a weak TH agonistic activity for TBBPA in the absence of T3, whereas a TH antagonistic activity was found for TBBPA at higher concentrations in the presence of T3, showing that the P. nigromaculatus assay is effective for detecting TH signaling disrupting activity. Importantly, we observed non-monotonic dose-dependent disrupting activity of TBBPA in the presence of T3, which is difficult to detect with in vitro reporter gene assays. Overall, the developed P. nigromaculatus assay can be used to screen TH signaling disrupting activity of environmental chemicals with high sensitivity.

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Molecular characterization and mRNA expression of ribosomal protein L8 in Rana nigromaculata during development and under exposure to hormones

Cited by 16 publications

References 37 publications

Low concentrations of dihydrotestosterone induce female‐to‐male sex reversal in the frog Pelophylax nigromaculatus

Low concentrations of dihydrotestosterone induce female‐to‐male sex reversal in the frog Pelophylax nigromaculatus

Tetrabromoethylcyclohexane affects gonadal differentiation and development in the frog Pelophylax nigromaculatus

A screening assay for thyroid hormone signaling disruption based on thyroid hormone-response gene expression analysis in the frog Pelophylax nigromaculatus

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