In vitro induction of sperm nucleus decondensation by cytosol from mature toad eggs

Abstract

An in vitro assay was performed on the cytosol from oocytes of the toad, Bufo bufo japonicus, to examine the cytoplasmic activity that induces sperm nucleus decondensation (SND). When the sperm, pretreated for 15 min with 0.05% Triton X-100, were incubated in cytosol obtained from mature eggs (18 h post-hormone treatment: PHT) 30–60 min after activation, their nuclei elongated and swelled markedly to take a spherical shape between 1 and 2 h after incubation. The treatment of such detergent-treated sperm nuclei with dilute HC1 significantly enhanced this process of nuclear decondensation. The SND activity was retained in the pellet after centrifugation at 105,000g for 1 h. It was completely inhibited by EGTA (1 mM) and serine protease inhibitor (1 mM), though not at all by soybean trypsin inhibitor (1 mg/ml). The cytosol from unactivated eggs at 18 h PHT had hardly any SND activity, but was induced to exhibit a strong activity when 10 mM Ca²⁺ was added to the extraction medium. No SND activity was observed in the cytosol from full-grown ovarian oocytes (0 h PHT), oocytes at the first meiotic metaphase (11 h PHT), or activated oocytes at 18 h PHT from which the germinal vesicle had been removed before the hormone treatment. Electron-microscopic observations revealed that detergent- and acid-treated sperm had completely lost their nuclear envelopes, but, after exposure to active egg cytosol, the decondensed nuclei were surrounded by continuous membranous envelopes. These results point to the importance for the formation of swollen nuclei in fertilized eggs of the membrane system in mature egg cytoplasm that may be activated by Ca²⁺ at the time of egg activation.