Characterization of vitellogenin concentration in male fathead minnow mucus compared to plasma, and liver mRNA

Abstract

The objective of this study was to characterize vitellogenin (VTG) protein in male fathead minnow (Pimephales promelas) mucus compared with more conventional measures in plasma and mRNA isolated from liver. To assess the intensity and duration of changes in mucus VTG concentrations, male fathead minnows were exposed to 17α-ethinylestradiol (EE2) for 7 days with a subsequent depuration period of 14 days. The experiment was conducted in a flow-through system to maintain a consistent concentration of EE2 at a nominal EC₅₀ concentration of 2.5 ng/L and high concentration of 10 ng/L as a positive control. Mucus, plasma and liver were sampled at regular intervals throughout the study. Relative abundance of vtg mRNA increased after 2 days of exposure and returned to control levels after 4 days of depuration. VTG protein concentration displayed similar induction kinetics in both mucus and plasma, however, it was found to be significantly increased after 2 days of exposure using the mucus-based assays and 7 days with the plasma-based assay. Significantly elevated levels of VTG were detected by both assays throughout the 14-day depuration period. The elimination of the laborious plasma collection step in the mucus-based workflow allowed sampling of smaller organisms where blood volume is limiting. It also resulted in significant gains in workflow efficiency, decreasing sampling time without loss of performance.

Keywords: Biomarkers; Biomonitoring; ELISA; Estrogenic compounds; Gene expression; RT-qPCR.

Fig. 1.

Concentration of vitellogenin (VTG) over time in mucus of male fathead minnows exposed to A,B Control (0.00 ng/L, black circle), A High (5.48 ng/L, orange triangle), or B Low (1.33 ng/L, blue square) EE2 for 7 days followed by a 14-day depuration in clean water. Each point represents the mean with vertical lines displaying standard deviation (n = 8–10). Vertical dashed gray line indicates the beginning of the depuration period. Horizontal dotted line marks VTG (ng mL⁻¹) = 0. Asterisks indicate significant difference between the means of Control and High or Low within that day, determined by Welch’s ANOVA with Games-Howell post-hoc test (*p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001). Individual sample values may be found in Fig. S4.

Fig. 2.

Concentration of vitellogenin (VTG) over time in plasma of male fathead minnows exposed to A,B Control (0.00 ng/L, black circle), A High (5.48 ng/L, orange triangle), or B Low (1.33 ng/L, blue square) EE2 for 7 days followed by a 14-day depuration in clean water. Each point represents the mean with vertical lines displaying standard deviation (n = 3–5). Vertical dashed gray line indicates the beginning of the depuration period. Horizontal dotted line marks VTG (ng mL⁻¹) = 0. Asterisks indicate significant difference between the means of Control and Low or High within that day, determined by Welch’s ANOVA with Games-Howell post-hoc test (*p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001). Individual sample values may be found in Supplementary Fig. S5.

Fig. 3.

Expression of vitellogenin (vtg) over time in liver of male fathead minnows exposed to A,B Control (0.00 ng/L, black circle), A High (5.48 ng/L, orange triangle), or B Low (1.33 ng/L, blue square) EE2 for 7 days followed by a 14-day depuration in clean water. Each point represents the mean with vertical lines displaying standard deviation (n = 8 – 10, Day 21 n = 4). Vertical dashed gray line indicates the beginning of the depuration period. Asterisks indicate significant difference between the means of Control and Low or High within that day, determined by Welch’s ANOVA with Games-Howell post-hoc test (*p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001). Individual sample values may be found in Supplementary Fig. S6.