Henri Goudeau

Ascidian eggs block polyspermy by two independent mechanisms: One at the egg plasma membrane, the other involving the follicle cells

Many ascidians live in clumps and usually release sperm before the eggs. Consequently, eggs are often spawned into dense clouds of sperm. Because fertilization by more than a single sperm is lethal, ascidians have evolved at least two successive blocks to polyspermy: the rapid release of a glycosidase that inhibits sperm binding to the vitelline coat (VC) and a subsequent change in membrane potential that prevents supernumerary sperm–egg fusion. This paper shows that (1) these two blocks can be uncoupled by the use of suramin, and (2) most of the glycosidase appears to be from the follicle cells, which are accessory cells on the outside of the egg VC. Phallusia mammillata eggs initially bind numerous sperm but, after the glycosidase is released, only a few additional sperm bind. Intact eggs in 20 μM suramin release glycosidase, but the electrical response is inhibited; sperm swim actively and bind to the VC but fail to penetrate. Suramin treatment is completely reversible; intact eggs exhibit the electrical response an average of 11 minutes after the drug is washed out. Sperm must contact the follicle cells before passing through the VC; eggs with the VC removed and fertilized in the presence of 20 μM suramin show the electrical response 35% of the time, thus VC removal enhances sperm entry. Like the intact eggs, 100% of the naked eggs respond electrically to fertilization after the drug is washed out. Follicle cells that are isolated by calcium magnesium free seawater and then returned to complete seawater release N‐acetylglucosaminidase activity in response to sperm. Thus, these eggs have two blocks to polyspermy that operate in sequence: an early first block resulting from enzymatic modification of the VC by N‐acetylglucosaminidase released primarily from follicle cells and a second electrical block operating at the egg plasma membrane level and requiring sperm–egg fusion. Mol. Reprod. Dev. 48:137‐143, 1997.

The resumption of meiotic maturation of the oocyte of the prawn Palaemon serratus is regulated by an increase in extracellular Mg2+ during spawning

Abstract The resumption of meiotic maturation in the oocyte of the prawn Palaemon serratus was triggered by exposure to either natural or artificial seawater. The stages of meiotic maturation were observed in whole mounted oocytes. Resumption of meiosis depended on the presence of external Mg 2+ but not on external Ca 2+ . The kinetics of the completion of meiotic maturation stimulated by artificial seawater with or without Ca 2+ were identical. The threshold concentration of external Mg 2+ required for the resumption of meiosis was ⩾ 15 m M . This result is of physiological interest, since this Mg 2+ concentration was higher than the Mg 2+ concentration in blood from ripe female prawns (10 m M ). Therefore, Mg 2+ might be a natural factor controlling meiotic maturation in the prawn oocyte. Addition of ionophore A 23187 to artificial seawater also induced meiotic maturation; the stimulation by A 23187 required external Ca 2+ but not external Mg 2+ .

Tributyltin impedes early sperm-egg interactions at the egg coat level in the ascidian Phallusia mammillata but does not prevent sperm-egg fusion in naked eggs

Abstract The impact of tributyltin (TBT), was investigated on the occurrence of early gamete contacts and the functioning of the ionic channels of unfertilized or fertilized egg of Phallusia mammillata. TBT significantly inhibited the Na+ current of unfertilized oocytes up to 60%, in both a dose (1–10 μM) and time (30 min to 8 h) dependent manner, while it moderately affected the Ca2+ and K+ currents. Also TBT induced a statistically significant dose-dependent decrease in the percentage of eggs that responded electrically to fertilization (from 100 to 33%, respectively, for 0.1–3 μM TBT). As the sperm concentration increased, the dose-response curve of the percentage of eggs that responded electrically to fertilization shifted towards higher TBT concentrations which did not affect the capability for sperm to fertilize eggs. The effects of TBT were weakly reversible, depending on both TBT concentrations and the duration of incubation in TBT solutions. TBT (5 μM) prevented early sperm egg contacts (5×107 sp ml−1) at the vestment level of intact eggs, but did not lower the percentage of electrical responses in naked eggs. Nevertheless, it had a deleterious effect on the transduction mechanism of the sperm signal into the egg or, more directly, on the sperm-activated channels of the egg membrane. This was evidenced by naked eggs displaying strongly truncated electrical responses, although they were penetrated by sperm-nuclei. Finally, N-acetylglucosamine, an intrinsic vitelline coat (VC) monosaccharide involved in the sperm binding to VC, added (100 mM) to 5 μM TBT in natural seawater, in order to compete with the pollutant, did not reduce the deleterious effect of TBT on the early gamete contacts at VC level.

Electrical and morphological responses of the lobster egg to fertilization

Abstract The electrophysiological and morphological characteristics of fully grown immature and fertilizable mature oocytes of the European lobster Homarus gammarus are described. In fully grown immature oocytes with a centrally located germinal vesicle, the resting potential was −54 ± 1.5 mV (n = 32) and the membrane was K+ selective. Fully grown immature oocytes with a peripheral germinal vesicle whose Em was −40 ± 1.5 mV (n = 11), were predominantly permeable to K+, and slightly permeable to Cl−. In mature oocytes arrested at first meiotic metaphase the plasma membrane was selectively permeable to Cl−, and Em was −32 ± 2 mV (n = 16). Mature oocytes were inseminated in vitro with sperm from the thelycum or female sperm receptacle. Insemination instantaneously triggered a sustained hyperpolarization that corresponded to the fertilization potential, and was caused by increased membrane permeability to K+. Concomitant with this electrical response, the oocyte, which before insemination had been in first metaphase, resumed its meiotic maturation. One hour after insemination, the nucleus of the penetrating spermatozoon was in the egg cortex.

Extracellular Mg2+ induces a loss of microvilli, membrane retrieval, and the subsequent cortical reaction, in the oocyte of the prawn Palaemon serratus

Surface changes induced by sea water were analyzed in the ovulated oocyte of the prawn Palaemon serratus. They depended on the presence of external Mg2+ but not on external Ca2+ alone. Increasing external Mg2+ from 0 mM to 30 mM stimulated first a progressive disappearance of preexisting microvilli, which was over within 30 min of incubation. This is correlated with membrane removal via internalization of coated vesicles, ascertained by observations of endocytosis of an extracellular fluid-phase marker and by measurement of a diminution in membrane capacitance (Cm). Thirty-five minutes after sea water contact, the prawn oocyte underwent a cortical reaction independent of fertilization. It consists in a heavy exocytosis of ring-shaped elements, leading to the deposition of a thick capsule, and requiring a threshold Mg2+ concentration of greater than or equal to 10 mM and at least a 3-min incubation with Mg2+. Concurrently, the values of the membrane capacitance (Cm) and conductance (Gm) increased about 2 and 10 times their initial values, respectively. The calcium ionophore ionomycin, added to Mg(2+)-free artificial sea water, stimulated the cortical reaction with requirement of external Ca2+. Other divalent cations (Mn2+, Zn2+, Co2+, Ni2+, Cd2+) instead of Mg2+, induced the cortical reaction, but Ba2+, Sr2+, and La3+ did not. When eggs are fertilized, the cortical reaction takes place in two steps, the first being a discrete exocytosis of a foamy material and the second always involving ring-shaped elements.

MERCURIC IONS IMPAIR THE FERTILIZATION POTENTIAL, THE RESUMPTION OF MEIOSIS, THE FORMATION OF MALE PRONUCLEUS, AND INCREASE POLYSPERMY, IN THE EGG OF THE ASCIDIAN PHALLUSIA MAMMILLATA

In the sea squirt Phallusia mammillata, fertilization occurs externally in the seawater surrounding the adult. The impact of mercuric ions, one of the most toxic environmental pollutants, was investigated on the functioning of the ionic channels of unfertilized or fertilized egg membrane and on the associated egg morphological events occurring at fertilization. Hg2+ ions in the submicromolar range affected in a dose-dependent manner (range 0.0625 to 2 μM) the functioning of voltage-dependent Na+ and Ca2+ channels pre-existing in the unfertilized oocyte membrane. These channels are also sequentially involved in the first step of the egg electrical response to fertilization (i.e., the fertilization potential) and were impaired by Hg2+. Mercuric ions also strongly inhibited the further operating sperm-dependent channels of this fertilization potential. The two highest concentrations used (1 and 2 μM) greatly reduced the number of eggs that responded electrically and, as a consequence, diminished the percentage of fertilized eggs that resumed meiosis. Also as a consequence of the impairment by Hg2+ ions of the egg electrical response to fertilization, the electrical block to polyspermy became less efficient and the proportion of polyspermic eggs increased. Finally, the percentage of eggs containing a male pronucleus over the total number of sperm-penetrated eggs statistically diminished in a dose-dependent manner under the effect of Hg2+ ions. J. Exp. Zool. 278:255–272, 1997.

External Mg2+ is required for hyperpolarization to occur in ovulated oocytes of the prawn Palaemon serratus

Immediately after dissection, the ovulated oocyte of the prawn Palaemon serratus had a resting potential Em of −42 ± 2 mV and a membrane resistance Rm of 15 ± 5 MΩ; the membrane was more permeable to Cl− than to K+. The oocyte spontaneously hyperpolarized and Em gradually reached −70 mV 20–30 min after removal of the oocyte from the female, due to increased membrane permeability to K+. However, the hyperpolarization occured only if Mg2+ was present in the seawater; external Ca2+ was not required. Long-term incubation without external Mg2+ depolarized the membrane and increased membrane resistance. After preincubation in Mg2+-free ASW, oocytes transferred to standard artificial seawater (ASW) transiently hyperpolarized and then repolarized, before gradually hyperpolarizing to a sustained value of −62 ± mV. The respective roles of external Mg2+ and fertilization in eliciting the electrical response of the prawn egg at natural spawning are discussed.

Influence of a transepithelial NaCl gradient on the moulting cycle, keratinization and active sodium transport of isolated frog skin cultured with or without aldosterone

SummaryIsolated frog skins were maintained, in organ culture in a modified Ussing chamber for up to 9 days with or without transepithelial NaCl gradient and aldosterone. Without gradient, (86 mM NaCl Ringer in the mucosal compartment and Wolf and Quimby amphibian medium culture in the serosal compartment), the structural organization of the epithelium, moulting cycle, keratinization and active sodium transport were similar to those observed before culture. In the absence of gradient, aldosterone slightly intensified keratinization and was necessary to maintain a high rate of active sodium transport. In the presence of a transepithelial ionic gradient (5 mM NaCl Ringer in the mucosal compartment and Wolf and Quimby amphibian medium culture in the serosal compartment), skins evolved towards the ultimate stage of the moulting cycle and the rate and the degree of keratinization were strongly enhanced. Aldosterone obviously promoted overlapping of the last two phases of the cycle and further intensified keratinization. It also strikingly raised active sodium transport. The epithelia of trypsinized skins, stripped of their stratum corneum and stratum granulosum were able to restructure themselves in culture. In this newly formed epithelium, the former regular structural organization was not preserved and the moulting cycle was no longer distinguishable. Moreover, when there was no transepithelial gradient, the keratinization process slowed down considerably. The presence of a gradient (28 mM NaCl Ringer in the mucosal compartment and Wolf and Quimby culture medium in the serosal compartment) promoted keratinization and led to the formation of cornified layers, which were sometimes detached from the underlying epithelial layers.