2 results
Nicotinic acetylcholine receptors of the neuronal type occur in the plasma membrane of sea urchin eggs
- Pedro I. Ivonnet, Edward L. Chambers
-
- Article
- Export citation
-
The addition of acetylcholine (ACh, 100 μl of 10 μM) to the bath in the vicinity of unfertilised sea urchin eggs (Lytechinus variegatus) suspended in sea water (SW) abruptly depolarises the membrane potential (Vm) of the eggs from the resting value of approximately −70 mV. This results in the firing of the egg's action potential, followed by partial repolarisation. Similar addition of ACh to eggs voltage clamped at −70 mV induces an inward current of abrupt onset with peak amplitude of −1.26 ± 0.20 nA (SE, n = 81). When the eggs are clamped at a Vm more positive than −70 mV, the peak amplitude of the ACh-induced inward current decreases, becoming 0 at a clamped Vm of approximately −20 mV. Further positive shift of the Vm fails to cause reversal of the current. Oocytes clamped at −70 mV exhibit similar inward current responses following application of ACh. Since ACh stimulates both nicotinic and muscarinic receptors (nACh-R and mACh-R, respectively), the effects of exposing eggs to the agonists and antagonists for each type of receptor were examined. For unfertilised eggs clamped at −70 mV the application of 100 μM (−)-nicotine hydrogen tartrate, an agonist of the nACh-R, induces an inward current response similar to that elicited by 10 μM ACh, but of smaller peak amplitude. In contrast, the application of (+)-muscarine chloride, an agonist of the mACh-R, fails to induce any response. Antagonists of the nACh-R inhibit either the neuronal type of nACh-R or the skeletal muscle type of nACh-R. The effect of the antagonists on the amplitudes of the ACh-induced inward current response was determined by supervising individual eggs clamped at −70 mV with the desired antagonist dissolved in SW, followed by the addition of 100 μl of 10 μM ACh in the vicinity of the egg. Mecamylamine chloride, an antagonist of the neuronal nACh-R at a concentration of 1 μM, markedly decreases the response to ACh, while at a concentration of 10 μM the response to ACh is abolished. Hexamethonium chloride, another inhibitor of the nACh-R of the neuronal type, also diminishes the ACh-induced response, but at a concentration of 10 μM the response is not completely abolished. Exposure of eggs to α-bungarotoxin, an antagonist of the skeletal muscle nACh-R at concentrations up to 250 nM for periods of 30 min, has no effect on the ACh-induced response. The effects of two antagonists of the mACh-R, atropine sulphate and QNB (R-(−)-3-quinuclidinyl benzilate) were also examined. Exposure of eggs to 1 μM atropine does not affect the ACh-induced response, but at concentrations of 10 μM atropine the amplitude of the ACh-induced inward current is significantly reduced. The exposure of eggs to QNB, a highly specific antagonist of the mACh-R, at concentrations up to 50 nM, has no effect on the ACh-induced response. Consequently, the likely explanation for the inhibitory effect of atropine is that at high concentrations atropine cross-reacts with the nACh-R. These findings reveal the presence in unfertilised sea urchin eggs of an ACh-R resembling the neuronal nACh-R. No evidence could be obtained that these receptors have a role in sperm entry, activation of the egg, or early development.
Stages leading to and following fusion of sperm and egg plasma membranes
- Frank J. Longo, Susan Cook, David H. McCulloh, Pedro I. Ivonnet, Edward L. Chambers
-
- Article
- Export citation
-
The site of gamete interaction of electrophysiologically recorded Lytechinus variegatus eggs, fixed with osmium tetroxide (O5O4) and/or glutaraldehyde (GTA) at varying intervals after the onset of the increase in membrane conductance induced by an attached sperm, has been examined by high-voltage and conventional transmission electron microscopy. Although GTA and a GTA-O5O4 mixture iduced different electrical responses, specimens prepared with the two fixatives were ultrastructurally similar. In specimens observed within 5 s of the change in conductance, the acrosomal process projected through the vitelline layer and abutted the egg plasma membrane. A conspicuous layer of bindin surrounded the acrosomal process and connected the sperm to the egg's vitelline layer. In a fortuitous specimen fixed within 4 s following the change in conductance, the area of contact between the gamete plasma membranes possessed a trilaminar structure that separated the egg's and sperm's cytoplasms. The morphology of this area of contact was consistent with previously proposed intermediates of membrane fusion. Five to six seconds after the change in conductance, the sperm was connected to the egg via a narrow cytoplasmic bridge that consisted of the former acrosomal process and a projection of the egg cortex. The region of the bridge midway between the fused gametes was encircled by dense material that marked the site of sperm-egg fusion. Gamete interactions in which the activation potential was recorded (unclamped egg) were comparable in time and ultrastructure to events taking place in voltage-clamped eggs except for one major difference. Intact cortical granules (one to three) were observed beneath the tip of the incorporating sperm in unclamped eggs fixed following the onset of the activation potential, whereas all cortical granules dehisced in clamped eggs.