OVERRIPENING OF OVULATED EGGS IN GOLDFISH, CARASSIUS AURATUS L

Abstract

The morphological, physico-chemical and endocrinological changes associated with egg overripening in goldfish, Carassius auratus, were investigated. Overripening occurred within 24 h after ovulation at 23°C. During the course of overripening, significant morphological and physico-chemical changes were observed in the eggs and ovarian fluid. Three stages could be distinguished microscopically and histologically. Morphological changes in eggs included the aggregation of the cytoplasm with the cortical alveoli (CA) and oil droplets (OD) at the animal pole and the partial breakdown of CA. The overripe eggs appeared more transparent, and also became bigger due to hydration. The chorion became thinner and surface changes were noted, e.g. the completel withdrawal of the plugs from the pores or the partial recession of the plugs into the pores. The time course of overripening was established based on various parameters. Overripening started at around 3 h after ovulation when the chorion thickness decreased. Although only a few changes in the eggs and no changes in the ovarian fluid were observed during the early stage of overripening (6 h), there was a significant drop in fertilization and hatching rates. By 12 h (late stage) and 24 h (final stage) more significant changes occurred in the eggs, i.e. marked reductions in protein, ATP, energy charge (EC) and chorion thickness, as well as marked changes in the elements (Na+, K+, Ca++ and Mg++). The ovarian fluid at 24 h showed marked increases in protein and Na+ levels as well as in osmolality and volume/amount. These results implicate changes of egg permeability during overripening. Steroid hormone changes were also studied in both serum and ovarian fluid. In the serum, a significant decline in progesterone (P) and 17a, 20ß-dihydroxy-4-pregnen-3-one (17, 20ß-P) was observed during the period of egg overripening, while in the ovarian fluid only 17, 20ß-P decreased significantly; these changes or the reduced ratios of P and 17, 20ß-P in relation to E2 showed a good correlation with the time course of overripening. Histological examination revealed that the degeneration of the postovulatory follicles (POFs) coincided well with overripening and the decline of the steroids. The effect of P on overripening in vivo was studied by immersing ovulated fish in P or 17, 20ß-P for 12 h at 23°C. Based on fertilization rates, treatment with P (0.05 ppm) and to a lesser extent with 17, 20ß-P (0.05 ppm) was able to delay overripening for a few hours. There were significant increases in the ATP and EC levels of eggs from fish immersed in P. The effect of P on overripening was also investigated in vitro by incubation of ovulated eggs in ovarian fluid together with anti-P at 23°C. Based on fertilization rates, this treatment resulted in a faster rate of overripening after 6 h of incubation, suggesting a direct role of P on ovulated eggs. Incubation of ovulated eggs in M199 and Ringer's (but not in ovarian fluid) resulted in autoactivation, i.e. formation of a perivitelline space as a result of cortical alveoli breakdown. Addition of P and/or E2 to M199 and Ringer's did not prevent or alleviate this phenomenon. Thus it was not P and/or E2 but other substance(s) in the ovarian fluid (e.g. proteins) which prevented the breakdown of the cortical alveoli in the eggs. The above results, taken together, suggest that the decline of P in the ovarian fluid (by itself or in relation to other steroids, e.g. E2), as a result of the degeneration of postovulatory follicles, causes, or at least contributes to the overripening of ovulated eggs in the ovarian fluid. How this is brought about by the hormone change is not clear, but it is clear that overripening is accompanied by marked morphological and physico-chemical changes suggestive of a drop in energy charge and an increase in permeability.