Quantification of serum levels of precursors to vitelline envelope proteins (choriogenins) and vitellogenin in estrogen treated masu salmon, Oncorhynchus masou

Abstract

Previously two precursors to vitelline envelope proteins, choriogenin H (Chg H) and choriogenin L (Chg L), were identified in masu salmon, Oncorhynchus masou, and specific antisera against these two proteins were generated in rabbits. In this study, two methods of immunoassay have been developed using these specific antibodies: single radial immunodiffusion (SRID) and enzyme-linked immunosorbent assay (ELISA). Non-competitive sandwich ELISAs for Chg H and Chg L were designed using digoxigenin-labeled antibodies and purified Chgs as assay components. The working range of the ELISAs was 1–128 and 2–256 ng/ml for Chg H and Chg L, respectively. Using these immunoassays and a chemiluminescent immunoassay for vitellogenin (Vg), the changes in these three estrogen-responsive proteins were measured in the serum of masu salmon after treatment with various doses of estradiol-17β (E 2). The changes in serum levels of Chgs and Vg in male fish differed according to the E 2 dose. When fish were given a 5 mg/kg body weight (BW) of E 2, Vg was induced to a greater extent than Chgs. By contrast, Chg levels were higher than that of Vg after a 10 μg/kg BW of E 2 injection. A similar trend was seen in the response time to exogenous E 2. Serum Chgs were induced from 8 h after E 2 injection and reached a peak of about 5 μg/ml at 24 h. Although Vg was not detected until 8 h after E 2 injection, its levels remained considerably low at around 0.03 μg/ml, even after 24 h. Chg H was more sensitive than was Chg L to 1 μg/kg BW of estrogen: the long-term exposure of fish to E 2 showed that Chg H could be induced from a lower dose of E 2 than could Chg L. Taken together, these results suggest that the serum levels of Chg H, Chg L, and Vg in masu salmon are regulated by circulating levels of E 2.