Abstract:

Exposure to 17β-estradiol (E2) has been shown to influence the regulation of multiple signaling pathways during development. Disruption of signaling events during early development can lead to malformations such cardiac defects. In this study we looked the expression of two genes involved in cardiac development, hand2 and lrrc10 as well as the expression of gper and cardiac development at various stages of development in zebrafish. Zebrafish embryos were exposed to concentrations of E2 (0.1μM, 2μM, 5μM, 7μM and 8μM) at 2 hours post fertilization (hpf) with subsequent measurements of lrrc10, hand2 and gper transcripts at 12hpf, 28hpf and 76hpf. Concentration dependent alteration in the expression of lrrc10, hand2 and gper was observed at 12hpf and 76hpf, but not at 28hpf. Expression of these genes were also altered after exposure to the GPER agonist G1. Expression was correlated with the formation of cardiac edema as well as other developmental abnormalities indicating a role of GPER in cardiac development.

Suggested Citation:

Graciel Diamante. 2017. Effects of 17β-estradiol (E2) on the development of zebrafish embryos. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N7P848X9

Publications:

Diamante, G., Menjivar-Cervantes, N., Leung, M. S., Volz, D. C., & Schlenk, D. (2017). Contribution of G protein-coupled estrogen receptor 1 (GPER) to 17β-estradiol-induced developmental toxicity in zebrafish. Aquatic Toxicology, 186, 180–187. doi:10.1016/j.aquatox.2017.02.024

Purpose:

To further understand the mechanisms of toxicity of estrogenic compounds during development.

Data Parameters and Units:

S009 folder: Survival data; Treatment; Number alive (counts); Number dead (counts); Total (counts); Survival ratio (alive/total); Percent survival (alive/total x 100); Average of percent survival; Standard deviation (stdev); Standard error (sterror) S010 folder: Amplification data at 76 hours post fertilization (hpf) S011 folder: Amplification data at 12 hours post fertilization (hpf) S012 folder: Amplification data at 28 hours post fertilization (hpf) Treatment= The solution the zebrafish embryos were exposed to. (Control-0.1% ethanol and the Test compound was 17beta-estradiol (E2) at different concentrations) Set=Replication number given Ct(threshold cycle)= is the average CT obtained from the raw data generated for each replicate for that gene. Genes of interest: GPER= G-protein coupled estrogen receptor gene, Lrrc10= Leucine Rich Repeat Containing 10 gene, Hand2= Heart And Neural Crest Derivatives Expressed 2 gene, Housekeeping gene EFa or ELF= elongation factor, VTG= vitellogenin Average Ct Hand2= average of the threshold cycle for Hand2 for all the sets Average Ct Lrrc10= average of the threshold cycle for Lrrc10 for all the sets Average Ct GPER= average of the threshold cycle for GPER for all the sets Average Ct Efa= average of the threshold cycle for EFa for all sets Average Ct VTG = average of the threshold cycle for VTG for all sets Standard deviation Lrrc10= standard deviation of the threshold cycle for Lrrc10 for all the sets Standard deviation Hand2= standard deviation of the threshold cycle for Hand2 for all the sets Standard deviation GPER= standard deviation of the threshold cycle for GPER for all the sets Standard deviation EFa= standard deviation of the threshold cycle for EFa for all the sets 2-Delta Delta Ct= (The CT of the gene of interest for a specific subtracted of the CT of EFa for that set) subtracted (from the average Ct of gene of interest for the control group subtracted from the average CT of EFa for the control group) Average 2-Delta Delta Ct= average of all the 2-Delta Delta Ct for that treatment group Standard deviation 2-Delta Delta Ct= standard deviation of all the 2-Delta Delta Ct for that treatment group Standard Error 2-Delta Delta Ct= standard of all the 2-Delta Delta Ct for that treatment group over the number of replicates Fold change= Ratio of the Average 2-Delta Delta Ct over the control group (0.1% ethanol) S013 folder: Deformities data; Treatment; Cardiac deformities (counts); Total (counts); No deformities (counts); Ratio of cardiac deformities (deformed/total); Percent of cardiac deformities (deformed/total x 100); Average of percent cardiac deformities; Standard deviation (st dev); Standard error S014 folder: cAMP data; Raw plate reads; Concentration (ug/mL); Treatment; Concentration of 50% dilution (mg/mL); Concentration of stock (mg/mL); Temperature (deg C) S018 folder: survival and deformity data

Methods:

qPCR, Calcium measurements, cAMP, abnormality analysis

Instruments:

Biorad thermocycler

Individual Files: