Embryonic development of medaka fish egg after nanosecond pulsed electric field application

Abstract

The paper describes the effects of 50 to 300 nanosecond pulsed electric field on the embryonic development of the medaka fish egg (Oryzias latipes) in a low and high conductivity medium. In the recent years it has been reported that applying short (less than 100 ns) pulses increased the possibility of electric field interactions with subcellular structures, which leaded to secondary cellular events, such as temporal increase in cell membrane permeability and induction of apoptosis. The goal of the current study was to find the effects of short pulsed electric field in-vivo and during embryo development. A pulsed power modulator using a magnetic compression circuit (MPC) was employed to generate 0.5 to 20 kV pulses with 50 to 300 nanosecond pulse durations. Input voltage and current were measured by using an oscilloscope and a current monitor. Fertilized eggs of strain d-rR medaka were used. The age of the experimental eggs were new laid of 1 and 6 hour post fertilization (hrPF), and 1 day and 2 days old. In each experiment, a single medaka egg (about 1.2 mm diameter) was set at the middle of a 2 mm or 4 mm cuvette and a single electric pulse was applied. After the experiments the eggs were observed under a microscope until they hatched or died. A fluorescent plasma membrane integrity indicator, propidium iodide (PI), was used to study electroporative uptake kinetics of the embryo cells after the electric pulse exposure. By applying 300 ns electric pulses, extensive damage of eggs were observed immediately after pulse application. For shorter 50 ns width pulses and low electric field, delayed hatching consistent with electric field subcellular interaction was observed, whereas stronger electric field affected the eggs immediately after the pulse and those eggs could not survive and died a few days later. To study the effects of pulsed electric field to other internal structures fluorescein isothiocyanate (FITC) was injected inside the yolk sphere or blastomere of 1 hrPF eggs. For FITC injection, a micro-injection system was used, which can deliver nano-liter order of FITC without damaging the eggs. After pulse application, the experimental eggs were monitored under a fluorescent microscope and damages to eggs' internal structures were studied.