Yasuaki Yoshimoto

Time Sequence of Early Events in Fertilization in the Medaka Egg

The time sequence of early events in fertilization was examined in eggs of the medaka Oryzias latipes. The mean time after insemination required for sperm attachment to the egg surface through the micropyle depended on sperm concentrations. It was 3 ± 1 sec with a range from 1 to 6 sec after insemination when concentration of spermatozoa was high (about 2 × 108/ml at 23°–25°C). The mean time from sperm attachment until cessation of its movement on the egg surface was 4 ± 1 sec with a range from 1 to 9 sec. Small cortical alveoli at the animal pole region within 15 μm of the sperm attachment point began to undergo exocytosis 9 ± 0.3 sec (range 5–16 sec) after sperm attachment. The velocity at which the exocytosis wave propagated increased from the earliest initiation point of exocytosis up to the 100 μm area, and became constant at about 12 μm/sec from 100 μm to 500 μm from the sperm attachment point. The present results suggest that at the time of fertilization in the fish egg, exocytosis of small cortical alveoli in the area about 15 μm away from the sperm attachment point occurs simultaneously.

Cytoplasmic Ca2+ release induced by microinjection of Ca2+ and effects of microinjected divalent cations on Ca2+ sequestration and exocytosis of cortical alveoli in the medaka egg

Intracellular release of Ca2+ by microinjection of Ca2+ was analyzed by measuring the luminescence of aequorin loaded in eggs of the medaka (Oryzias latipes). Microinjection of Ca2+ into the cortical cytoplasm induced propagative waves of cytoplasmic Ca2+ release and exocytosis of cortical alveoli initiated at the injection point. The Ca2+ wave was initiated with a time lag after some was sequestered at the region of the microinjection. Microinjection of Mg2+ or Mn2+ failed to trigger Ca2+ release and exocytosis. When the aequorin-loaded eggs were inseminated after microinjection of Mg2+, Mn2+, or Co2+ into a restricted region of the vegetal hemisphere, the wave of Ca release was propagated through the injected region toward the vegetal pole, but neither Ca sequestration (fall in Ca-aequorin luminescence) nor exocytosis occurred at the area of cortex where the eggs were injected with these divalent cations. These results suggest that a significant period is required to induce Ca2+ release from cytoplasmic stores by the increased Ca2+ concentration and that both the phenomena of Ca2+ release and Ca sequestration are involved in the process of exocytosis.

Observation of Intracellular Ca2+ with Aequorin Luminescence

Publisher Summary This chapter focuses on the studies on Ca 2+ observation carried out by the aequorin– (video-intensified microscopes) VIM method. It also illustrates the principle of VIM and describes its modified version. The principal part of VIM is the VIM camera consisting of a two-dimensional photon-counting tube and an ordinary vidicon video camera. The optical image from a microscope formed on the photocathode is converted into an electron beam that is then accelerated and focused by electric lenses onto a two-stage microchannel plate. Since the success in extracting aequorin from luminescent jellyfish by Shimomura et al. , aequorin luminescence has been used by many investigators to determine intracellular Ca 2+ concentration, taking advantage of its specificity to Ca 2+ and sensitivity up to the micromolar concentrations of Ca 2+ . However, early studies were limited by insufficient sensitivity because a submicromolar concentration of Ca 2+ was often required for the determination. This problem has been partly solved by the advent of semisynthetic aequorins with various synthetic lumiphores. Samples free of toxicity can be chosen from various semisynthetic aequorins. The absolute Ca 2+ concentration can be determined from the ratio of the intensities of luminescence at two definite wavelengths of a kind of semisynthetic aequorin. In addition, improvements in the two-dimensional light-detection technique and the image-processing technique have made possible to determine the distribution of submicromolar Ca 2+ concentrations in single cells.

Activation of Xenopus Eggs by Cynops Sperm Extract is Dependent upon Both Extra-and Intra-Cellular Ca Activities

Abstract When unfertilized Xenopus eggs were treated by Cynops sperm extract in 10% Steinbergs solution (SB), eggs membranes hyperpolarized to about −37 mV and then depolarized to elicit a positive-going potential amounting to about +34 mV. The eggs underwent cortical contraction and resumption of meiosis. Activation of eggs in various external solutions indicates that the hyperpolarization is due mainly to opening of Na channels, but the positive-going potential is due to Cl channels on the eggs plasma membranes. Since the activation was inhibited by CdCl2, CoCl2, or NiCl2 as well as by amiloride, Ca influx through Ca channels is necessary for the activation by the sperm extract. A propagative intracellular Ca release was induced not only by Cynops sperm, but also by their sperm extract. Injection of BAPTA or heparin into the eggs completely inhibited activation, indicating that egg activation requires an intracellular Ca release dependent upon receptors for inositol 1,4,5-trisphosphate.

Nuclear Involvement in Localization of the Initiation Site of Surface Contraction Waves in Xenopus Eggs

The initiation site of surface contraction waves (SCWs) was examined in fertilized, parthenogenetically activated and enucleated Xenopus eggs after either rotation through 90° off the vertical axis or injection of colchicine. In enucleated eggs, SCWs always started from a top site of the egg under all conditions examined. In fertilized or activated eggs, SCWs started, depending on the experimental conditions, from either the sperm entry point, the animal pole region located sideward or the top site of the egg. Histological examinations of fertilized and activated eggs revealed that the nucleus was in most cases positioned close to the initiation site of SCWs under various experimental conditions. It is suggested from these results that the egg cytoplasm has an intrinsic capability of causing the surface to generate SCWs, and that the nucleus is generally involved in localizing the initiation site of SCWs in fertilized or activated Xenopus eggs. A possible mechanism for localizing the initiation site of SCWs in Xenopus eggs is proposed.