Sperm-egg fusion in the sea urchin is blocked in Mg²⁺-free seawater

Abstract

SummaryMagnesium ions as well as calcium ions are required for successful fertilisation in sea urchins. In the absence of Mg2+ spermatozoa attached to the egg plasma membrane, their acrosomal processes passing through the vitelline envelope, but could not enter the egg cytoplasm (Sano et al, Dev. Growth Differ. 22, 531–41,1980). Such an individual spermatozoon was observed microscopically to resume entry into the egg immediately after the addition of a sufficient amount of Mg2+ to the surrounding medium. Neither any change in membrane potential nor an increase in intracellular Ca2+ concentration of the egg was observed after insemination in the absence of Mg2+, although both could be observed after the addition of Mg2+. The sperm heads did not show fluorescence when attached to the surface of an egg previously microinjected with mithramycin A in Mg-free seawater, indicating that there was no connection between the sperm and the egg. Therefore, occurrence of fertilisation potential must be a post-fusional event. These results suggest that Mg2+ are indispensable for fusion between the sperm acrosomal membrane and the egg plasma membrane.

Development of calcium releasing activity induced by inositol trisphosphate and cyclic ADP‐ribose during in vitro maturation of sea urchin oocytes

“…Internalized sperm were detected according to the method of Mohri et al (1994). Oocytes or eggs, to which DNA staining fluorescent dye, mithramycin A (Sigma), had been injected to a final concentration of 100 µM, were inseminated at a 1000-fold dilution, 5 min later fixed with 1% glutaraldehyde (Wako Pure Chemicals, Oosaka, Japan) for 30 s, followed by washing with FSW, and observed under a BH-2 fluorescence microscope (Olympus).…”

Regulation of intracellular calcium release during fertilization in sea urchin eggs

“…It is difiBcult to precisely remove ASW by centrifiigation (Ciapa and Maggio, 1993) fi-om samples at 10 sec interval during the first 2 min of fertilization. Although sea urchin eggs can be fertilized in a Ca^^-fi-ee medium by using acrosome-reacted sperm (Schmidt et al, 1982), there is a requirement for in ASW during sea urchin fertilization (Sano and Mohri, 1976;Mohri et al, 1994). Preliminary trials showed a low and inconsistent fertilization rate using ASW with low amounts of Ca^ and Thereafter we determined a reduction of Ca^"^ and Mg^* contents in ASW in half was sufficient for high fertilization rate with minimal interference of the assay by chelating the residual and Mg^* with additions of 10 mM EGTA and 40 mM EDTA in 2 N KOH for neutralization of phosphoric acid.…”

“…Eggs in a 1% egg suspension were mixed with an equal amount of Ca^^ and Mg^^-free ASW consisting of 545 mM NaCl, 11.4 mM KCl and 6 mM NaHCCb, pH 8 (Mohri et al, 1994), to make a 0.5% egg suspension in a solution with only half of the Ca^^ and Mg^" contents in normal ASW (l/2CaMgASW). Eggs were then incubated with or without 20 jiM U73122 in a 0.5% egg suspension for 5 min and reconstituted to 7.5% before insemination.…”