21 research outputs found
Sexing Frogs by Real-Time PCR: Using Aromatase (cyp19) as an Early Ovarian Differentiation Marker
Most anurans have no identified sex-markers; therefore, alternative
methods for identification of early changes in sex
ratios are required. In this study, Lithobates sylvaticus and Silurana
tropicalis tadpoles were sampled at different developmental
stages covering the entire process of sex differentiation.
Three candidate genes known to be involved in sex differentiation
in other vertebrate species were selected to
develop a method to identify phenotypic sex in frogs: cytochrome
p450 aromatase (cyp19) , forkhead box L2 (foxl2) and
the cytochrome 17-alpha-hydroxylase/17,20 lyase (cyp17) .
Cloning of these genes revealed nucleotide identity values
ranging between 75–97% when compared to other amphibian
species. Gene expression of cyp17, cyp19 and foxl2 in
L. sylvaticus adult gonads and gonad-mesonephros complex
(GMC) of tadpoles was analyzed by real-time RT-PCR. Results
showed clear sexually dimorphic patterns in the expression of the 3 genes. Our analysis reveals that GMC gene expression levels of cyp19 alone can be used as a robust predictor
of phenotypic sex in L. sylvaticus tadpoles. In addition, we
validated this method measuring cyp19 mRNA levels in S.
tropicalis GMCs. We propose measuring cyp19 as a tool to
study the effects of chemical contaminants (including endocrine
disrupting compounds) on amphibian gonadal development
and sex ratios in the future
Sexing frogs by real-time PCR : using aromatase (cyp19) as an early ovarian differentiation marker
Most anurans have no identified sex-markers; therefore, alternative methods for identification of early changes in sex ratios are required. In this study, Lithobates sylvaticus and Silurana tropicalis tadpoles were sampled at different developmental stages covering the entire process of sex differentiation.Three candidate genes known to be involved in sex differentiation in other vertebrate species were selected to develop a method to identify phenotypic sex in frogs: cytochrome p450 aromatase (cyp19), forkhead box L2 (foxl2) and the cytochrome 17-alpha-hydroxylase/17,20 lyase (cyp17). Cloning of these genes revealed nucleotide identity values ranging between 75–97% when compared to other amphibian species. Gene expression of cyp17, cyp19 and foxl2 in L. sylvaticus adult gonads and gonad-mesonephros complex(GMC) of tadpoles was analyzed by real-time RT-PCR. Results showed clear sexually dimorphic patterns in the expression of the 3 genes. Our analysis reveals that GMC gene expression levels of cyp19 alone can be used as a robust predictor of phenotypic sex in L. sylvaticus tadpoles. In addition, we validated this method measuring cyp19 mRNA levels in S.tropicalis GMCs. We propose measuring cyp19 as a tool to study the effects of chemical contaminants (including endocrine disrupting compounds) on amphibian gonadal development and sex ratios in the future
Supplementary Material for: Sexing Frogs by Real-Time PCR: Using Aromatase <b><i>(cyp19) </i></b>as an Early Ovarian Differentiation Marker
Most anurans have no identified sex-markers; therefore, alternative methods for identification of early changes in sex ratios are required. In this study, <i>Lithobates sylvaticus</i> and <i>Silurana tropicalis</i> tadpoles were sampled at different developmental stages covering the entire process of sex differentiation. Three candidate genes known to be involved in sex differentiation in other vertebrate species were selected to develop a method to identify phenotypic sex in frogs: cytochrome p450 aromatase <i>(cyp19)</i>, forkhead box L2 <i>(foxl2)</i> and the cytochrome 17-alpha-hydroxylase/17,20 lyase <i>(cyp17)</i>. Cloning of these genes revealed nucleotide identity values ranging between 75–97% when compared to other amphibian species. Gene expression of <i>cyp17,</i><i>cyp19</i> and <i>foxl2</i> in <i>L. sylvaticus</i> adult gonads and gonad-mesonephros complex (GMC) of tadpoles was analyzed by real-time RT-PCR. Results showed clear sexually dimorphic patterns in the expression of the 3 genes. Our analysis reveals that GMC gene expression levels of <i>cyp19</i> alone can be used as a robust predictor of phenotypic sex in<i> L. sylvaticus </i>tadpoles. In addition, we validated this method measuring <i>cyp19</i> mRNA levels in <i>S. tropicalis </i>GMCs. We propose measuring <i>cyp19</i> as a tool to study the effects of chemical contaminants (including endocrine disrupting compounds) on amphibian gonadal development and sex ratios in the future