Vitellogenin and vitellogenesis in greenback flounder Rhombosolea tapirina

The overall aim of this study was to obtain knowledge about the yolk precursor, vitellogenin (Vtg) and the process of vitellogenesis in female greenback flounder (Rhombosolea tapirina), including purification and characterization of Vtg; development of a specific Vtg assay; induction of Vtg by l 7P-estradiol (E2) in vivo and in vitro; relationship between endocrine regulation of vitellogenesis and oocyte growth in mature females; and the possible role of Vtg feedback on ovarian steroid production. Vtg purified from the plasma ofE2-treated male animals by gel filtration chromatography had an estimated molecular weight (MW) of~ 540 kD, and resolved into three bands with estimated MW of 152, 102, 80 kD after SDSp AGE. Western blotting demonstrated that the Vtg consisted of one subunit (152 kD). A polyclonal antibody against the 152 kD band developed from sheep, was used to establish-an indirect antibody-capture competitive enzyme-linked immunosorbent assay (ELISA). The validation of the ELISA was confirmed by the parallelism between the standard Vtg curve and serial dilutions of plasma from vitellogenic females, but no cross-reaction was found with the plasma of males, or plasma from a range of other species. In Ez-treated fish, detectable concentrations of plasma Vtg were first found at 48 h, and reached a peak at 96 h post-injection, and increased as the E2 dose increased, with a threshold of~ 0.1 mg E2 Kg body weight -1. Multiple injections of E2 increased the absolute concentrations of Vtg and extended the peak Vtg up to 288 h, but did not change the threshold dose. The time-course and dose response for induction of Vtg by E2 in the flounder was similar to that seen in other species, however, plasma concentrations of Vtg were generally lower than those found in other teleosts. Oocyte size-frequency distributions from mature females showed that this species has multiple clutch group synchronous type of oocyte development. But that reproductive development is not synchronized within the population. Consistent with this observation, there were no significant seasonal variations in gonadosomatic index (IG), hepatosomatic index (IH), or plasma concentrations of Vtg, E2 and testosterone (T). Significant increases in IG , IH and plasma concentrations of Vtg, E2 and T were observed in vitellogenic fish, and in fish undergoing final maturation. Plasma concentrations of Vtg and E2 rose steadily across oocyte sizes from 100 to 450 μm, and both reached a concentration plateau at oocyte sizes of around 450 μm. In contrast, plasma concentrations of T showed no marked increase until oocytes grew beyond 400 μm. Isolated vitellogenic follicles were incubated with human chorionic gonadotropin (hCG), dibutyryl cyclic AMP (dbcAMP) and gonadal steroid precursors T, 17- hydroxyprogesterone (17P) and androstenedione (A) in the presence or absence of Vtg. High concentrations ofVtg suppressed the production of E2 and T, and the effect appeared to operate at several levels along the steroidogenic pathways. These observations combined with findings of other studies indicate that Vtg might regulate its own production by modulating ovarian steroid synthesis