Voltage dependence of the [Ca2+](i) oscillations system, in the Mg2+ -stimulated oocyte of the prawn Palaemon serratus.

By voltage-clamp technique and simultaneous [Ca2+](i)measurements, we studied the modifications, induced by changes in membrane voltage, in the pattern of the [Ca2+](i)oscillation period, displayed by the Mg2+-stimulated oocyte of the prawn Palaemon serratus. When the Mg2+-stimulated oocytes were voltage clamped at 0mV, they developed a [Ca2+](i)signal with a more pronounced oscillatory pattern than that obtained on unclamped oocytes. Indeed, they displayed a first peak followed by a series of sharp [Ca2+](i)transients and a prominent [Ca2+](i)oscillatory plateau. By contrast, oocytes voltage clamped at - 60mV showed a first peak followed by a stable high [Ca2+](i)level forming a long continuous plateau devoid of oscillations. By using caged InsP3, we established that the ER InsP3 receptor is not voltage sensitive. Paradoxically, we showed the voltage sensitivity of the Mg2+ receptor-signal transduction system which is more reactive to Mg2+ ions at -60mV than at 0mV. Using different calmodulin inhibitors of the PM CA pump such as trifluoperazin (100microM), W-7 (50microM) and calmidazolium (50microM), we suppressed the [Ca2+](i)oscillatory pattern in oocytes voltage clamped at 0mV. From these results we propose that this special voltage-dependent oscillatory system could be regulated by a significant involvement of the electrogenic PM CA pump.

Widely meshed autograft associated with cultured autologous epithelium for the treatment of major burns in children: report of 12 cases.

This is a retrospective study of the combination of widely meshed autograft and autologous cultured keratinocytes. We used this method faced with the lack of allogenic skin, as an alternate to the Cuono method. Twelve children suffering extensive burn injury (deep burns of 60%+/-16 of the total body surface) underwent this grafting procedure. The surgical treatment consisted of an early surgical excision, with an immediate coverage by autografts as much as possible. When cultured epithelium was available, a large mesh autograft was applied and covered with cultured epidermis sheets during the same operative procedure. The rate of take was of 84% (+/- 12). No secondary graft loss was observed. This means of coverage appeared reliable and resistant. On average, this method allowed the epidermization of 30% (+/-9) of the total body surface of the children. The average hospital stay of the children was 64+/-20 days. All the children recovered to lead a normal life. The school delay after rehabilitation is one year. This technique is an alternative to Cuono's method when allografts are missing. The combination of autograft and autologous cultured epidermis sheets appeared more effective than one of these techniques applied alone, as if the suggested coupling induced a synergy.

Depletion of intracellular Ca2+ stores, mediated by Mg2+-stimulated InsP3 liberation or thapsigargin, induces a capacitative Ca2+ influx in prawn oocytes.

By voltage clamp technique and intracellular calcium measurements, we recorded in prawn oocytes simultaneous [Ca2+]i and ionic current changes stimulated by external Mg2+. The [Ca2+]i response consists of an oscillation period followed by a second state of sustained [Ca2+]i level. The oscillation period successively comprises a first [Ca2+]i peak, a series of [Ca2+]i transients, and a [Ca2+]i oscillatory plateau respectively concurrent with an initial transient outward K+Ca current, an inward Na+Ca current, and a final K+Ca outward current. By using inhibitor (heparin) or sensitizers (thimerosal or caffeine) of calcium release ER channels, and caged InsP3, we established that InsP3 is the sole second messenger releasing Ca2+ from intracellular stores. By sequential substitutions and reapplications of external Ca2+, and using econazole (50 microM), a Ca2+ influx inhibitor, we documented Ca2+ influx during the [Ca2+]i oscillatory plateau. The intracellular Ca2+ store was depleted with thapsigargin (75-350 nM) in Ca2+-free ASW. Reapplication of external Ca2+ evoked a rise in [Ca2+]i, indicating a store-dependent capacitative Ca2+ influx, correlated with a K+Ca outward current increase. No measurable Ca2+ release-activated Ca2+ current (Icrac) could be detected, but was indirectly demonstrated using the sensitivity of the K+Ca channels to [Ca2+]i. We propose that the involvement of external Ca2+, in the physiological [Ca2+]i response of prawn oocytes to external Mg2+, consists of a store-dependent capacitative Ca2+ influx.

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 microM 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 microM 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.

Role of the tricuspid annulus and the eustachian valve/ridge on atrial flutter. Relevance to catheter ablation of the septal isthmus and a new technique for rapid identification of ablation success.

BACKGROUND: Typical atrial flutter (AFL) results from right atrial reentry by propagation through an isthmus between the inferior vena cava (IVC) and tricuspid annulus (TA). We postulated that the eustachian valve and ridge (EVR) forms a line of conduction block between the IVC and coronary sinus (CS) ostium and forms a second isthmus (septal isthmus) between the TA and CS ostium. METHODS AND RESULTS: Endocardial mapping in 30 patients with AFL demonstrated atrial activation around the TA in the counter-clockwise direction (left anterior oblique projection). Double atrial potentials were recorded along the EVR in all patients during AFL. Pacing either side of the EVR during sinus rhythm also produced double potentials, which indicated fixed anatomic block across EVR. Entrainment pacing at the septal isthmus and multiple sites around the TA produced a delta return interval < or = 8 ms in 14 of 15 patients tested. Catheter ablation eliminated AFL in all patients by ablation of the septal isthmus in 26 patients and the posterior isthmus in 4. AFL recurred in 2 of 12 patients (mean follow-up, 33.9 +/- 16.3 months) in whom ablation success was defined by the inability to reinduce AFL, compared with none of 18 patients (mean follow-up, 10.3 +/- 8.3 months) in whom success required formation of a complete line of conduction block between the TA and the EVR, identified by CS pacing that produced atrial activation around the TA only in the counterclockwise direction and by pacing the posterior TA with only clockwise atrial activation. CONCLUSIONS: (1) The EVR forms a line of fixed conduction block between the IVC and the CS; (2) the EVR and the TA provide boundaries for the AFL reentrant circuit; and (3) verification of a complete line of block between the TA and the EVR is a more reliable criterion for long-term ablation success.

External Mg2+ triggers oscillations and a subsequent sustained level of intracellular free Ca2+, correlated with changes in membrane conductance in the oocyte of the prawn Palaemon serratus.

We have provided evidence that external Mg2+ induces correlated changes in [Ca2+]i and membrane current or potential in prawn oocytes without any requirement of fertilization, using the fluorescent Ca2+ indicator Ca green dextran and voltage and current clamp methods. Replacement of Mg2+-free ASW with standard (40 mM Mg2+)ASW triggered a diphasic [Ca2+]i response consisting of an oscillation period followed by a second state of sustained [Ca2+]i level devoid of oscillation, lasting about 70 min and up to 3 hr, respectively. In contrast, oocytes maintained for a long time in Mg2+-free ASW showed no changes in [Ca2+]i which remained at a basal concentration of about 0.2 microM. The simultaneous records of [Ca2+]i and membrane or current changes showed that the oscillation period started with a first [Ca2+]i peak (about 1.7 microM) and was correlated with an initial transient membrane hyperpolarization or a transient initial outward current peak. The first [Ca2+]i peak was followed by a slow decrease in [Ca2+]i followed by a series of [Ca2+]i transients, concurrent with a slow depolarization of the membrane or a related inwardly directed current. The oscillation period ended with an oscillatory plateau of [Ca2+]i (about 0.6 microM) lasting 49.6 +/- 4.5 min, the onset of which was concomitant with a final membrane hyperpolarization or a related final outward current. A continuous contact between external Mg2+ and the oocyte membrane was necessary to maintain the program of [Ca2+]i and membrane conductance changes. The sources of Ca2+ mobilization are of internal origin for both the first peak and the subsequent series of [Ca2+]i oscillations, and are mainly of external origin for the oscillation plateau and for the second state of sustained [Ca2+]i level. The first and second step of the cortical reaction occurred during the oscillatory plateau and the second state of sustained [Ca2+]i level, respectively.

Evidence by a voltage clamp study of an electrically mediated block to polyspermy in the egg of the ascidian Phallusia mammillata.

Eggs of the ascidian Phallusia mammillata were voltage clamped (from -100 to +60 mV) and inseminated with a low or heavy sperm concentration. From inseminations with low sperm concentration (1 x 10(6) sp/ml), we found that fertilization currents occurred between -100 and +40 mV: they were always inward and displayed an analogous pattern whatever the clamped voltage. We established that the percentages of inseminated eggs that produced a fertilization current varied as a function of the clamped voltage. These percentages were not statistically different from 100% at clamped voltages between -100 and -30 mV, they decreased to 68 and 56% at clamped Vm of -10 and 0 mV, respectively, but were not statistically different from 0% at clamped Vm between +10 and +40 mV. We never obtained any egg electrical response at a clamped voltage of +50 mV. Almost all eggs (96%) which responded electrically were penetrated by one or several spermatozoa. These eggs were resuming meiosis (81 to 50%) at values of clamped Vm between -100 and 0 mV, respectively. At clamped Vm between +10 and +50 mV, the percentages of eggs resuming meiosis were not statistically different from 0. These results indicate that in P. mammillata eggs, the occurrence of an electrical response is voltage dependent and consequently that the initial depolarizing shift of the fertilization potential constitutes a fast block to polyspermy. However, in this species, the sperm penetration is not voltage dependent, since it occurred at clamped Vm from -100 to +40 mV. On the other hand, when eggs were clamped from -100 to +60 mV and inseminated with a heavy sperm concentration (2 x 10(7) sp/ml), the curves expressing, respectively, the percentages of eggs which responded electrically, the percentages of eggs which were penetrated by one or several spermatozoa, and the percentages of eggs resuming meiosis, as functions of the clamped Vm, were shifted by approximately 35 mV toward more positive voltages, compared to the corresponding curves obtained from eggs inseminated with a low sperm concentration. This last result means that the critical value of the membrane potential which characterizes the electrical block to polyspermy is dependent on the sperm concentrations used for inseminations.

In the egg of the ascidian Phallusia mammillata, removal of external Ca2+ modifies the fertilization potential, induces polyspermy, and blocks the resumption of meiosis.

The fertilization potential of the Phallusia mammillata egg has been found to consist, in natural or standard artificial sea water (NSW and ASW), of an initial sperm-triggered rapid depolarization comprising two distinct successive components, followed by a phase of membrane depolarization comprising a plateau and two series of membrane potential oscillations. These oscillations occur during meiotic divisions and they end just before formation of the second polar body. In the present work, the effects of removing external Ca2+ on the fertilization potential time course on the rate of polyspermy and on the resumption of meiosis of the P. mammillata egg were explored. Eggs inseminated in Ca(2+)-free ASW usually responded by several successive sperm-induced electrical responses strikingly different from that obtained in standard ASW: removing external Ca2+ during insemination and the initial depolarizing shift (free (Ca2+), 5-11 microM in Ca(2+)-free ASW) and during the subsequent period of the electrical responses (free (Ca2+), 0.2 microM in Ca(2+)-free ASW), completely suppressed both the second component of the initial depolarizing shift, the plateau, and membrane oscillations of the depolarization phase, did not impair sperm entry, but always blocked the resumption of meiosis. Under these conditions eggs were polyspermic (91%, n = 22, c.l. 69-99%). Lastly, only a short period of contact (approximately 20 sec at minimum) with a physiological concentration of external Ca2+ (12 mM) at the onset of the fertilization potential was necessary for the egg to resume meiosis and then to undergo complete embryonic development. These results indicate that in the P. mammillata egg, the second component of the initial depolarizing shift involves voltage-gated Ca2+ channels. The possibility is discussed that external Ca2+ ions might contribute to the activation of the P. mammillata egg by providing a source for an increase of (Ca2+)i and/or for the reloading of the Ca2+ internal stores.

Successive electrical responses to insemination and concurrent sperm entries in the polyspermic egg of the ctenophore Beroe ovata.

In the ctenophore Beroe ovata, action potentials and sperm-induced egg electrical responses were recorded, in unfertilized and in vitro inseminated eggs, respectively. The unfertilized egg had a resting membrane potential of -65.6 +/- 1.3 mV and a membrane resistance of 1.45 +/- 0.09 M omega. When depolarized beyond a threshold of about -20 mV, the membrane displayed an action potential with a fast phase and then a peak reaching 38 +/- 0.6 mV, followed by a positive declining plateau which lasted about 20 sec and ended by a biphasic repolarization of the membrane. The action potential was mainly Ca(2+)-dependent and to a lesser extent, Na(+)-dependent. Upon one or several inseminations, monospermic eggs generated one fertilization potential and polyspermic eggs generated several sperm induced egg electrical responses. These sperm-induced egg electrical responses were composed of a voltage-gated action potential elicited by sperm-egg contact, resembling the action potential triggered by a depolarizing current, followed by a true sperm-gated depolarizing plateau which lasted about 60 sec and was mainly Na(+)-dependent. The number of electrical responses displayed by each egg corresponded to the number of male pronuclei detected in its cortex. An electrical response occurred for all egg membrane potentials ranging from -78 to +16 mV. In addition, the histogram giving the number of eggs in each class of egg characterized by a number of sperm entries indicated that each sperm entry constituted an independent process. However, when we compared the effectiveness of the in vitro inseminations during electrophysiological measurements and the in vivo inseminations under simulated natural conditions, the results showed two predominant classes of fertilized eggs, with one and two male pronuclei in the cortex respectively. Consequently, although our overall results exhibit features characteristic of polyspermy, they do not yet preclude the possibility that the eggs of the ctenophore B. ovata might be monospermic, or might, under natural planktonic conditions, display a small number of sperm entries.

The fertilization potential and associated membrane potential oscillations during the resumption of meiosis in the egg of the ascidian Phallusia mammillata.

The fertilization potential in Phallusia mammillata consisted of an initial rapid depolarization. This initial sperm-triggered depolarization was followed by a phase of membrane depolarization which was of either long or short duration, depending on the eggs. When of long duration, the phase of membrane depolarization was divided into two periods: the first one began with a plateau (Em = +20.2 +/- 1.1 mV; duration = 1.7 +/- 0.14 min) which was followed by a series of membrane potential oscillations (n = 3.1 +/- 0.25) lasting 2.4 +/- 0.2 min. The second period also began as a plateau (Em = approximately 0 mV; duration = 3.40 +/- 0.20 min) which was followed by a series of oscillations (n = 11.5 +/- 0.5) lasting 11.8 +/- 0.6 min, followed by a membrane repolarization. The second series of oscillations often continued rising from the resting potential value. In the eggs displaying a short duration of membrane depolarization, the second period of depolarization was shortened (lasting only 3.5 +/- 0.5 min) since it lacked the second plateau. In addition it displayed a smaller number of oscillations (n = 4.7 +/- 0.6). As a consequence of this shortening, the membrane repolarized sooner. After repolarization, the membrane displayed several potential oscillations that started from the repolarization level. Regardless of the length of the depolarized plateau phases, the total number of membrane oscillations and the time period during which they occurred were constant. Eggs displaying a long depolarization phase had 15.9 +/- 0.6 oscillations in a 19.5 +/- 0.6 min interval, while eggs having a short depolarization phase had 16.0 +/- 0.8 oscillations in a 18.1 +/- 0.3 min interval. The time period during which the potential oscillations occurred corresponded remarkably well with the time of the meiotic divisions: the formation of the first polar body was detected about 80 sec after the end of the first series of oscillations; the second polar body was extruded about 85 sec after the last membrane oscillation occurred.

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.

[Value of fibrin glue in surgery of patients with severe burns]. / Intérêt de la colle de fibrine dans la chirurgie du grand brûlé.

From January 1985 to May 1986, fibrin glue was used for graft sealing in 158 cases of our 200 skin grafts performed for the treatment of burns. When the graft area was less than 200 cm2, primary and complete healing was routinely observed. In the remainder, we noticed a higher quality of healing when fibrin glue was used compared to the other grafts. In 2 patients, infection of the wound was responsible for a total graft lysis which occurred immediately in the non-sealed grafts and was delayed in the sealed ones. Fibrin glue shortens skin graft healing time while it procures a better quality of life in patients with burns during in hospital stay. However the use of this healing-facilitating compound has to be limited to well-defined indications.

High titers of ecdysteroids are associated with the secretory process of embryonic envelopes in the european lobster.

The newly laid egg of the lobster Homarus gammarus is surrounded by a vitelline coat. Just after fertilization, a new subjacent envelope (2), originating from the cortical reaction, is deposited beneath the vitelline coat. In the course of embryonic development, five new coatings (envelopes 3 to 7) are secreted successively from the ectodermal embryonic cells. These will remain until hatching, freeing the mysis larva in concentric order without exuviation. The concentration of both the two major ecdysteroids (ponasterone A and 20-hydroxyecdysone) and their respective precursors (25-deoxyecdysone and ecdysone) were determined as a function of the secretory phase for three embryonic envelopes (2, 3 and 6). We determined that the secretory processes proceed in the presence of high titers of 20-hydroxyecdysone during the onset of envelope secretion and of ponasterone A in the last phase of secretion.

A long-lasting electrically mediated block, due to the egg membrane hyperpolarization at fertilization, ensures physiological monospermy in eggs of the crab Maia squinado.

In response to fertilization, the membrane potential (Em) of the crab egg hyperpolarizes from about -50 mV to about -80 mV in 400 msec. To establish whether this fast hyperpolarization is correlated with physiological polyspermy or conversely mediates an electrical block to polyspermy, we examined the morphological and electrophysiological characteristics of eggs from the crab Maia squinado. Fertilized naturally spawned eggs were found to be physiologically monospermic and their average Em was constant at -77 +/- 0.5 mV. To examine a possible electrical block ensuring this monospermy, unfertilized eggs were voltage clamped at various Em values ranging from +20 to -90 mV, inseminated, and examined morphologically. All eggs clamped at +20 to -65 mV responded by developing a fertilization current, If. It consisted of an outwardly directed K+ current in one or several steps, each caused by a single spermatozoon interacting with the egg membrane. The percentage of eggs clamped at values more negative than -65 mV, which responded at insemination by developing an If, decreased and dropped to 0 at -80 mV. This indicated that the membrane processes occurring during the contact between gametes and eliciting an electrical response by the egg membrane are voltage dependent. Further, the spermatozoon never penetrated into eggs voltage clamped at a Em between +20 and -60 mV and at voltages more negative than -75 mV. Em values between -65 and -75 mV were required for spermatozoon incorporation into the egg, indicating that sperm entry is also voltage dependent. It is proposed that the hyperpolarization of the egg membrane in response to fertilization constitutes a long-lasting electrical block to polyspermy in crab eggs.

Measurement of an intracellular pH rise after fertilization in crab eggs using 31P-NMR.

The effect of fertilization upon the intracellular pH, pHi, in crab ovulated eggs was examined by 31P-NMR. The pHi values were obtained from the chemical shift differences between the phosphoarginine PA resonance and the inorganic phosphate Pi resonance. The detection of the Pi peak was accomplished by Hahn spin-echo experiments in order to cancel the broad signal arising from phosphoproteins which overlaps the Pi signal. The average pHi of the unfertilized unactivated eggs was 6.55 and a rise of 0.12 pH unit occurred after fertilization.

A complex cortical reaction leads to formation of the fertilization envelope in the lobster, Homarus.

We have examined the formation of the fertilization envelope in the lobsters Homarus americanus and H gammarus. Oocytes were fixed for electron microscopy either in the ovary or following extrusion from the gonopore. Mature ovarian oocytes are surrounded by a coat (envelope 1), which is comprised of small electron-dense granules and structures resembling "bottlebrushes." At least part of this coat is synthesized by the follicle cells of the ovary. The cortex of ovarian oocytes contains four types of vesicles that we refer to as high-density vesicles (HDV), low-density vesicles (LDV), moderately dense vesicles (MDV), and ring vesicles (RV). Oocytes that were electrically extruded from the gonopore and fixed immediately had an envelope identical to that of ovarian oocytes. The cortex of gonopore oocytes contained the four types of vesicles found in ovarian oocytes. When unfertilized gonopore oocytes were allowed to incubate in sea water, the oocyte cortex appeared unaltered, but envelope 1 swelled and the bottlebrushes dispersed. When recently fertilized oocytes were fixed during natural spawning or following in-vitro fertilization, each type of vesicle was released in sequence from the cortex of the oocyte. The contents of the HDV and LDV appeared first in the perivitelline space, but their fate could not be determined at later times. The ring-shaped elements of the RV and the moderately electron-dense material of the MDV were released exocytotically somewhat later; these materials coalesced in the perivitelline space to form a new coat (envelope 2). Envelope 1 subsequently condensed to its original thickness and appeared firmly attached to envelope 2. Our results show that the fertilized lobster egg is surrounded by two discrete coats. The outer coat, which is formed in the ovary, undergoes a swelling/condensation cycle at spawning. The inner coat originates from a complex cortical reaction. Together these coats comprise the fertilization envelope of the lobster egg.

Mechanism of egg attachment stalk formation in the lobster, Homarus.

We have examined the formation of the egg attachment stalk in the lobsters Homarus americanus and H. gammarus. The formation of the stalk is similar in both species. Ovulated oocytes are surrounded by a single coat, envelope 1, composed of layers 1A and 1B. After passage through the gonopore and exposure to sea water, envelope 1 swells and becomes sticky. A second coat, envelope 2, forms between the oocyte and envelope 1 during a complex cortical reaction initiated after fertilization. Eggs pass over the ventral surface of spawning females to the region of the pleopods, where they stick by means of layer 1A to each other and to the ovigerous setae. Layers 1A and 1B are soft and pliable at this time. During egg attachment, the pleopods beat vigorously and cause envelope 1 to stretch and form attachment stalks. Beating probably also causes the attachment stalks to twist and wrap around the ovigerous setae. After the egg mass is secured to the ovigerous setae, envelope 1 of both the attachment stalk and egg coat condenses to form a tough material capable of securing the egg mass to the pleopods for intervals up to 16 months. After larvae hatch, portions of the egg coats and the attachment stalks are retained on the ovigerous setae until the female undergoes her next molt.

Structure of the egg funiculus and deposition of embryonic envelopes in a crab.

The newly laid egg of Carcinus maenas is attached to a maternal ovigerous seta by a funiculus which consists of the two superimposed vitelline envelopes 1a + 1b, highly stretched and concurrently showing important structural alterations. The funiculus is glued to the specialized seta merely owing to the strong adhesiveness of its external face comprising the outermost vitelline envelope 1a, without any added adhesive. The subjacent envelope 2, originated from the cortical reaction, is not involved in such a funiculus elaboration. In the course of the embryonic development, four new coatings are successively secreted from the ectodermal embryonic cells, underneath the (1a + 1b + 2) fertilization envelope or embryonic capsule. They will remain until hatching in this concentric order, thus giving evidence of successive embryonic moulting cycles, with apolysis but without exuviation. In addition, the successive secretory phases, regarding to the embryonic envelope elaborations, happen in presence of high concentrations of the ecdysteroid ponasterone A which might be involved consequently in such secretory processes.