Fertilized mouse eggs exhibit repetitive rises in intracellular Ca(2+) concentration ([Ca(2+)](i)) necessary for egg activation. Precise spatiotemporal dynamics of each [Ca(2+)](i) rise were investigated by high-speed Ca(2+) imaging during early development of monospermic eggs. Every [Ca(2+)](i) rise involved a Ca(2+) wave. In the first Ca(2+) transient, [Ca(2+)](i) increased in two steps separated by a "shoulder" point, suggesting two distinct Ca(2+) release mechanisms. The first step was a Ca(2+) wave that propagated from the sperm-fusion site to its antipode in 4-5 s (velocity, approximately 20 microm/s in most eggs). The second step from the shoulder to the peak was a nearly uniform [Ca(2+)](i) rise of 12-15 s. A slight cytoplasmic movement followed the Ca(2+) wave in the same direction and recovered in 25-35 s. These characteristics changed as follows, as Ca(2+) oscillations progressed during the second meiosis up to their cessation at the stage of pronuclei formation ( approximately 3 h after fertilization). (1) The duration of Ca(2+) transients became shorter. (2) The shoulder point shifted to higher levels and the first step occupied most of the rising phase. (3) The rate of [Ca(2+)](i) rise became greater and wave speeds increased up to 80-100 microm/s or more. (4) The transient cytoplasmic movement always resulted from the Ca(2+) wave, although its displacement became smaller. (5) The Ca(2+) wave initiation site was freed from the sperm-fusion or -entry site and eventually localized in the cortex of the vegetal hemisphere. Since the shift of the wave initiation site to the vegetal cortex is observed in fertilized eggs of nemertean worms and ascidians, this might be an evolutionarily conserved feature.

The mechanism of the elevation of intracellular free Ca2+ ([Ca2+]i) induced by a single sperm in eggs of the sea urchin Lytechinus variegatus was investigated. Simultaneous measurements of [Ca2+]i, and of the activation current, were carried out on eggs microinjected with Ca Green-1 or Ca Green dextran, and voltage clamped at -20 mV. The microinjection of 0.5 to 1.0 mg/ml heparin (MW 6000) or pentosan polysulfate (MW 3000), final intracellular concentration, causes a concentration-dependent inhibition in all parameters of the sperm-induced elevation of [Ca2+]i and the phase 2 calcium-activated cation current (Ip). For each: (1) the onset is delayed; (2) the rate of change is slowed; and (3) the peak amplitude attained is diminished. In some experiments at the higher concentrations, the microinjected polysulfates cause the complete suppression of the sperm-induced elevation of [Ca2+]i and Ip. The entry of multiple sperm overcomes the inhibitory effects of the polysulfates. Our data suggest that inositol 1,4,5-trisphosphate is the primary mechanism responsible for the sperm-induced release of Ca2+ from intracellular stores.

Behavioral traits of sperm are adapted to the reproductive strategy that each species employs. In polyandrous species, spermatozoa often form motile clusters, which might be advantageous for competing with sperm from other males. Despite this presumed advantage for reproductive success, little is known about how sperm form such functional assemblies. Previously, we reported that males of the coastal squid Loligo bleekeri produce two morphologically different euspermatozoa that are linked to distinctly different mating behaviors. Consort and sneaker males use two distinct insemination sites, one inside and one outside the female's body, respectively. Here, we show that sperm release a self-attracting molecule that causes only sneaker sperm to swarm. We identified CO2 as the sperm chemoattractant and membrane-bound flagellar carbonic anhydrase as its sensor. Downstream signaling results from the generation of extracellular H(+), intracellular acidosis, and recovery from acidosis. These signaling events elicit Ca(2+)-dependent turning behavior, resulting in chemotactic swarming. These results illuminate the bifurcating evolution of sperm underlying the distinct fertilization strategies of this species.

Hamster sperm extract (SE) possessing Ca2+ oscillation-inducing activity was microinjected into the peripheral or central region of mouse eggs, and the first increase in intracellular Ca2+ concentration ([Ca2+]i), together with the spread of fluorescence-labeled SE in the ooplasm, was investigated by imaging with confocal microscopy. Injection into the periphery always induced a Ca2+ wave that started from the injection site after a delay of 5 to 30 s depending on the concentration of SE. The diluted SE caused a wave of two-step [Ca2+]i rises, which was always observed at fertilization. Injection into the center could induce a radial Ca2+ wave with relatively high dose of SE, but lower dose of SE caused a [Ca2+]i rise after a longer delay which was initiated synchronously over the ooplasm or was preceded in a peripheral area. Injection of diluted SE remarkably prolonged the delay time and reduced the rate of [Ca2+]i rise. The critical concentration of SE needed to induce [Ca2+]i rise was significantly lower in the periphery. These results indicate that the sensitivity to SE is higher in the cortex. SE-induced [Ca2+]i rises were blocked by an antibody against the type 1 inositol 1,4,5-trisphosphate receptor (InsP3R). The cortex was substantially more sensitive to injected InsP3 induction of Ca2+ release than the center. It is suggested that the cortex of mouse eggs may involve a functionally specialized organization of InsP3Rs and Ca2+ pools in which a cytosolic sperm factor(s) could act upon sperm-egg fusion to cause Ca2+ release, leading to the Ca2+ wave at fertilization.

Most animal cell types regulate their cell volume after an osmotic volume change. The regulatory volume increase (RVI) occurs through uptake of NaCl and osmotically obliged water after osmotic shrinkage. However, apoptotic cells undergo persistent cell shrinkage without showing signs of RVI. Persistence of the apoptotic volume decrease is a prerequisite to apoptosis induction. We previously demonstrated that volume regulation is inhibited in human epithelial HeLa cells stimulated with the apoptosis inducer. Here, we studied signaling mechanisms underlying the apoptotic inhibition of RVI in HeLa cells. Hypertonic stimulation was found to induce phosphorylation of a Ser/ Thr protein kinase Akt (protein kinase B). Shrinkage-induced Akt activation was essential for RVI induction because RVI was suppressed by an Akt inhibitor, expression of a dominant negative form of Akt, or small interfering RNA-mediated knockdown of Akt1 (but not Akt2). Staurosporine, tumor necrosis factor-␣, or a Fas ligand inhibited both RVI and hypertonicityinduced Akt activation in a manner sensitive to a scavenger for reactive oxygen species (ROS). Any of apoptosis inducers also induced phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) in a ROS-dependent manner. Suppression of (ASK1) expression blocked the effects of apoptosis, in hypertonic conditions, on both RVI induction and Akt activation. Thus, it is concluded that in human epithelial cells, shrinkage-induced activation of Akt1 is involved in the RVI process and that apoptotic inhibition of RVI is caused by inhibition of Akt activation, which results from ROS-mediated activation of ASK1.