Richard A. Steinhardt

Intracellular calcium release at fertilization in the sea urchin egg

Abstract Fertilization or ionophore activation of Lytechinus pictus eggs can be monitored after injection with the Ca-sensitive photoprotein aequorin to estimate calcium release during activation. We estimate the peak calcium transient to reach concentrations of 2.5–4.5 μ M free calcium 45–60 sec after activation and to last 2 3 min, assuming equal Ca 2+ release throughout the cytoplasm. Calcium is released from an intracellular store, since similar responses are obtained during fertilization at a wide range of external calcium concentrations or in zerocalcium seawater in ionophore activations. In another effort to estimate free calcium at fertilization, we isolated egg cortices, added back calcium quantitatively, and fixed for observation with a scanning electron microscope. In this way, we determined that the threshold for discharge of the cortical granules is between 9 and 18 μ M Ca 2+ . Therefore, the threshold for the in vitro cortical reaction is about five times the amount of free calcium, assuming equal distribution in the egg. This result suggests that transient calcium release is confined to the inner subsurface of the egg.

Intracellular pH controls protein synthesis rate in the sea urchin egg and early embryo

Abstract Direct comparisons between intracellular pH and protein synthesis in the sea urchin egg and early embryo show that pH controls protein synthesis rate in a highly sensitive and reversible manner. The entire increase and maintenance of protein synthesis at fertilization or parthenogenetic activation could be accounted for by a permanent increase in intracellular pH. However, unfertilized eggs whose intracellular pH has been raised artificially by ammonia take at least 30 min longer to reach the rate of protein synthesis seen in fertilized eggs. This time lag for ammonia activation and the decrease in protein synthesis rate during mitosis suggest that other unknown factors can also influence protein synthesis rate during fertilization and early embryogenesis.

An analysis of the partial metabolic derepression of sea urchin eggs by ammonia: The existence of independent pathways☆

Incubation of unfertilized eggs in ammonia has been reported to initiate such late responses to fertilization as K+-conductance, DNA synthesis, chromosome condensation and cytoplasmic mRNA polyadenylation. It does not initiate such early responses as Na+-influx and the cortical reactions. We have further analyzed this metabolic derepression and find that ammonia activation does not result in the early respiratory burst and also does not initiate the late activation of Na+-dependent amino acid transport. Protein synthesis is increased, similar to that following normal fertilization. This indicates that augmentation of protein synthesis is causally linked neither to the earlier Na+-influx, cortical reactions, and respiratory burst nor to the later activation of amino acid transport. The temporal correlation between activation of transport and increased protein synthesis is therefore coincidental. The association between increased protein synthesis and establishment of K+-conductance was analyzed by abolishing K+-conductance through acidification of the sea water. This did not affect protein synthesis, indicating that K+-conductance and protein synthesis are also not causally linked. There is also no obligate link between protein synthesis and chromosome condensation. Incubation in low concentrations of ammonia results in increased protein synthesis but not chromosome condensation. Higher ammonia concentrations cause chromosome condensation but with no further increase in rate of protein synthesis. This suggests a concentration-dependent hierarchy of activation. These results are consistent with the concept that the late fertilization changes are not causally linked and proceed independently of each other. As we have not been able to disassociate the early changes, they may be obligately linked and dependent on each other.

Prevention of the cortical reaction in fertilized sea urchin eggs by injection of calcium-chelating ligands

Eggs from the sea urchin, Lytechinus pictus, were injected with either EGTA or EDTA, and were subsequently fertilized. EGTA prevented cortical vesicle discharge and formation of the fertilization membrane. EDTA had either no effect, or sometimes retarded the elevation of the fertilization membrane, or reduced the percentage of eggs with elevated membranes. Theoretical considerations lead to estimates of the probable effects of EGTA and EDTA on the internally released calcium which triggers the cortical reaction. Whether or not cytoplasmic calcium buffers are considered, it is concluded: (1) that normally several times the threshold calcium concentration for the cortical reaction is released into a subsurface space; (2) that if a rapidly-equilibrating high-affinity buffer is present, it is locally saturated by the calcium released internally; (3) the injected EDTA reduces the subsurface free calcium concentration normally reached to approximately threshold for the cortical reaction, while injected EGTA reduces the calcium concentration to below this threshold; and (4) a rise in the internal ionic calcium concentration is a necessary step in the activation of the cortical reaction at fertilization.

pH changes associated with meiotic maturation in oocytes of Xenopus laevis.

Abstract The pH-dependent control of the rate of protein synthesis in the fertilized sea urchin egg has suggested that pH may have an important role in cell activation at fertilization. We looked for similar changes of intracellular pH during meiotic maturation of the Xenopus oocyte. The basal pH of the oocyte is in the range 7.4–7.8. The higher values were found mostly in oocytes from animals with recent hormonal stimulation, suggesting a correlation with the elevated metabolism of such oocytes. Regardless of basal pH, progesterone-induced maturing oocytes alkalize an average of 0.18 pH unit. Beginning shortly before germinal vesicle breakdown, intracellular pH then decreases to near the original value. The same program is observed when maturation is induced by an injection of “maturation promoting factor.” Maturation is delayed or inhibited if intracellular pH is driven acidic. It is induced in the absence of progesterone by trimethylamine, a weak base which may act via an imposed alkalization. However, maturation still occurs when net alkalization is prevented. These data suggest that the alkalization during maturation is a form of metabolic “insurance” and that there may be both pH-dependent and pH-independent pathways for maturation. There is some evidence suggesting that pH changes are related to movement of other ions.

Intracellular calcium release and the mechanisms of parthenogenetic activation of the sea urchin egg.

Abstract Parthenogenetic activation of Lytechinus pictus eggs can be monitored after injection with the Ca-sensitive photoprotein aequorin to estimate calcium release during activation. Parthenogenetic treatments, including the nonelectrolyte urea, hypertonic sea water, and ionophore A23187, all acted to release Ca2+ from intracellular stores. Ionophore and urea solutions release Ca2+ from the same intracellular store as normal fertilization. This intracellular store can be reloaded after 40 min and discharged again. Hypertonic medium appears to release Ca2+ from a different intracellular store. Treatment with the weak base NH4Cl did not release intracellular Ca2+ but did result in a momentary Ca2+ influx if Ca2+ was present in the external solution. Ca2+ influx was not required for ammonia activation.

Proteolysis results in altered leak channel kinetics and elevated free calcium in mdx muscle.

SummaryResting free calcium levels ([Ca2−]i) are elevated in Duchenne human myotubes and mdx mouse muscle and myotubes which lack the gene product dystrophin at the sarcolemma. Increased net muscle protein degradation has been directly related to this elevated [Ca22+]i. The [Ca22+]i rise may result from increased calcium influx via leak channels, which have increased opening probabilities (Po) in dystrophic cells. Dystrophin, therefore, might directly regulate leak channel activity.In intact mdx soleus muscles, protein degradation was reduced to normal levels by leupeptin, a thiol protease inhibitor. In muscle homogenates, leupeptin also abolished calcium-induced increases in protein degradation. When mouse myotubes were cultured in the continuous presence of leupeptin (50 μm), the elevation in mdx resting [Ca22+]i was prevented. Leak channel Po increased with age in mdx myotubes, whereas leupeptin-treated mdx leak channel opening probabilities were always lower or equal to the Po for untreated normal myotubes.These results indicate that increased leak channel activity in dystrophic muscle results in elevated [Ca22+]i levels, but also suggest that dystrophin does not directly regulate channel activity. Instead the results suggest that proteolysis may be responsible for the altered gating of calcium leak channels. The resultant increased channel Po in turn elevates [Ca22+]i, which further increases proteolytic activity in a positive feedback loop, leading to the eventual necrosis of the muscle fibers.

Maturation of Xenopus oocytes. II. Observations on membrane potential.

Large, progesterone-responsive oocytes within their follicles have an average resting potential of about −25 mV. When manually dissected out of their follicles, most of these oocytes undergo a hyperpolarization over the next 30–60 min to values of about −60 to −80 mV. The relatively high negative membrane potentials previously recorded on dissected amphibian oocytes may thus be an artifact in the sense that such measurements do not reflect the electrical characteristics of the oocyte within the follicle. The available evidence indicates that the hyperpolarization reflects the activation of an electrogenic Na+,K+-transport process. One of the terminal events of oogenesis appears to be a suppression of the generation of the Na+,K+-transport process when oocytes are ovulated artificially (by dissection) or naturally. Continuous, long-term recordings on dissected oocytes reveal that a rather pronounced depolarization of the membrane potential together with an inflection in the recorded potential around the time of germinal vesicle breakdown takes place in the presence of progesterone. Recordings of oocytes within the follicle reveal similar changes, although reduced in absolute magnitude. In both cases, final membrane potentials of −10 to −15 mV are achieved. The electrophysiological changes which accompany the normal maturation process thus do not appear to be as pronounced as previously indicated.

Intracellular pH controls the development of new potassium conductance after fertilization of the sea urchin egg.

Abstract Fertilization of the sea urchin egg leads to a sequence of changes at the egg surface and the interior cytoplasm. Among these changes are the transient elevation of internal calcium levels, alkalization of the cytoplasm and development of new K+-conductance. In the series of experiments reported here, we separate the effects on potassium activation of the calcium release and the rise in the intracellular pH. The development of new K+-conductance was dependent on alkalization of the egg cytoplasm, and not on a rise of internal calcium levels. The effects of 2,4-dinitrophenol, N-ethylmalemide, antimycin A and oligomycin suggest that the maintenance of the alkaline internal pH of fertilized eggs appears to be dependent on membrane ATPase activity.

The relation between the increase in reduced nicotinamide nucleotides and the initiation of DNA synthesis in sea urchin eggs

Previous work has suggested that the activation of the sea urchin egg at fertilization is the result of a transient increase in intracellular free calcium and an increase in intracellular pH. We have investigated the absence of nuclear activation in incompletely activated eggs and have found a correlation between nuclear activation and the levels of total reduced nicotinamide nucleotides (NAD[P]H). Eggs activated with ammonia show a similar correlation: besides its action as a weak base in raising intracellular pH (which we conclude is insufficient to stimulate or maintain nuclear activation as judged by nuclear envelope breakdown or DNA synthesis), ammonia increases NAD(P)H. This increase is associated with the stimulation of 6-3H-thymidine incorporation into egg DNA. Removing ammonia decreased NAD(P)H, and tritiated thymidine incorporation ceases. We conclude that a critical level of NAD(P)H is essential to nuclear activation and that the increase of NAD(P)H at fertilization must be included with the increase in calcium and pH as a causal agent in development.