Fusion of lipid droplets in Japanese eel oocytes: Stage classification and its use as a biomarker for induction of final oocyte maturation and ovulation

The quality of eggs obtained from maturation-induced Japanese eel, Anguilla japonica, is unstable. One of the causes for low quality eggs is that females are artificially induced to ovulate at an inappropriate maturational status. Here we investigated the feasibility to use the morphological changes of lipid droplets in oocytes as a biomarker to show the optimum timing for injections of salmon pituitary extract (SPE) for priming and maturation-inducing steroid (MIS) for final oocyte maturation and ovulation. Various sizes of oocytes (700 to 1000 μm) were collected by cannulation from maturation-induced female eels during five days until ovulation. Morphology of the lipid droplets in these oocytes were classified into 10 stages mainly on the basis of their diameter, which increased with the progress of maturation as the droplets fused together. To assess the maturational status of each female, the lipid droplet stage was determined for 10 oocytes randomly chosen among the most advanced mode of developing oocytes and the median value was defined as the stage for the individual. Relationship between the lipid droplet stage at induction of ovulation and hatchability of the ovulated eggs was examined in 23 females, in which the hatching rate markedly fluctuated. Stages of females from which good quality eggs were obtained converged around stage 4 (five largest droplets, 55 to 70 μm) at priming with SPE, stage 6 (90 to 110 μm) at injection with MIS, and stage 7 (110 to 130 μm) at ovulation, while those of females from which low quality eggs were obtained showed distributions over broader ranges. Furthermore, 466 females were induced to mature and the lipid droplet stage at induction of ovulation was examined in 13 females from which high quality eggs (hatching rate >80%) were obtained. Most of the 13 females were at stage 3 (40 to 55 μm) or 4 when primed with SPE, at stage 6 when injected with MIS, and at stage 7 when they ovulated. These results suggest that the optimum stages for SPE priming, MIS injection, and ovulation are stages 3 to 4, 6, and 7, respectively, though the optimum stage for priming will be variable if the temperature and/or the time between priming and MIS injection is varied. We conclude that the lipid droplet stage can be used as a sensitive biomarker to show the optimum timing for inducing ovulation by hormonal treatments.