Comparative analyses of serum vitellogenin levels in male and female Reeves' pond turtles (Chinemys reevesii) by an immunological assay

Abstract

A quantitative immunological method was developed for measuring serum vitellogenin levels of Reeves' pond turtles (Chinemys reevesii) to investigate the effects of endocrine disruptors on the freshwater ecosystem. Vitellogenin was induced by injecting estradiol-17β into C. reevesii turtles (adult females, juvenile females, and males) and was purified from the turtle serum by EDTA-MgCl₂ precipitation followed by gel filtration. Using a polyclonal antibody raised against C. reevesii vitellogenin, an enzyme-linked immunosorbant assay was established. The detectable range, recovery of vitellogenin, and coefficient of variation in this assay were 0.0040–1.0 μg·ml⁻¹, 85.3–109% and 3.4–11.5%, respectively. This assay was also applicable for measurement of the concentrations of vitellogenins from other species, Japanese pond turtles (Mauremys japonica) and red-eared turtles (Trachemys scripta). The serum vitellogenin concentration of 131 C. reevesii turtles captured at a Japanese local river was measured by the assay. In females, vitellogenin ranged from 0.10 μg·ml⁻¹ to 15,000 μg·ml⁻¹ with two peaks, 0.10–1.0 μg·ml⁻¹ (juveniles) and 1,000–10,000 μg·ml⁻¹ (adults). However, in males, it ranged from 0.10 μg·ml⁻¹ to 0.60 μg·ml⁻¹, showing one peak, 0.10–0.20 μg·ml⁻¹. Therefore, if relatively high concentrations of vitellogenin are detected in males or juvenile females, it is suggested that they would have been exposed to xenobiotic estrogens.