Electrical and morphological responses of the lobster egg to fertilization

DEVELOPMENTAL

BIOLOGY

114,325-335

(1986)

Electrical and Morphological Responses of the Lobster Egg to Fertilization HENRI D&artement

GOUDEAU’ AND MARIE GOUDEAU

de Biologic, CEN Saclay, 91191G$-&r-Yvette Cedex; Labwabire de Zoologie Universite’ Pierre et Marie Curie, 75230Paris; U A. CNRS 686; and Station Marine, 29211Roscoff, France Received May 23, 1985;accepted October 23, 1985

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 f 1.5 mV (n = 32) and the membrane was K+ selective. Fully grown immature oocytes with a peripheral germinal vesicle whose E,,, was -40 + 1.5 mV (n = ll), 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 E,,, 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 o 1986 maturation. One hour after insemination, the nucleus of the penetrating spermatozoon was in the egg cortex. Academic

Press, Inc.

trical response of the egg tQ fertilization has a physiological significance specifically associated with internal In crabs, the fertilization potential consists of a rapid fertilization in crabs, or whether it is generally assoand sustained hyperpolarization of the egg plasma ciated with crustaceans. To answer this question, we unmembrane from -32 to -62 mV (Goudeau et aL, 1984; Goudeau and Goudeau, 1985). This hyperpolarization is dertook a study of the electrophysiological and morphodue to a change in the ionic permeability of the egg logical responses of decapod crustacean eggs, which are membrane, which becomes selective for K+, whereas be- fertilized in the external environment under natural fore insemination it was predominantly selective for Cl-. conditions. We chose the egg of the lobster which, apart A similar hyperpolarizing fertilization potential has not from Farmer’s opposite view (1974), is generally reported been reported in the eggs of other phyla (review in Hag- to undergo external fertilization (Bumpus, 1891;Herrick, iwara and Jaffe, 1979; Finkel and Wolf, 1980; Kline et 1909; Talbot, 1983). This paper reports our findings on the electrical charal, 1985), although transient hyperpolarizations have acteristics of fully grown immature and mature oocytes been seen in hamster (Miyazaki and Igusa, 1981, 1982), of the European lobster Homarus gammarus, as well as rabbit (McCulloh et aL, 1983), fish (Nuccitelli, 1980), and the egg fertilization potential and the associated mormouse oocytes (Jaffe et aZ.,1983). Furthermore, a small phological responses of the egg to in vitro insemination. (-10 mV) slow sustained hyperpolarization superimposed to the transient hyperpolarization occurs in the MATERIALS AND METHODS hamster egg (Igusa and Miyazaki, 1983;Igusa et al, 1983). The electrical response of the crab egg to fertilization Animals and Oocyte Collection was correlated with morphological events, thereby Female lobsters (H. gammarus) were obtained from showing that fertilization occurred. Under natural conditions, the early events in crab fertilization take place the fishery “La Langouste,” located near Roscoff in Britin the female genital duct and sometimes in the lumen tany. They were freshly captured and healthy. The feof the ovary (Binford, 1913;Spalding, 1942; Cheung, 1966; males were candled to evaluate ovarian development Goudeau, 1982; Anghelou-Spiliotis and Goudeau, 1982). (Hedgecock et al., 1978; Talbot, 1981a) and to select the The question then arises of whether this particular elec- ripest specimens. The females chosen were killed and dissected so that the morphology of the ovaries could be examined. The ovaries appeared to be in late stage 5 or ’ To whom requests for reprints should be addressed: Dkpartement stage 6 of development, according to the classification de Biologie, Service de Biophysique, CEN Saclay, 91191 Gif-Sur-Yvette Cedex, France. proposed by Aiken and Waddy (1980). The ovaries conINTRODUCTION

325

0012-1606/86 $3.00 Copyright All rights

0 1986 by Academic Press. Inc. of reproduction in my form reserved.

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tained large oocytes (1.2 to 1.6 mm in diameter), as well as medium-sized and small oocytes as reported by Talbot (1981a). We always studied the largest oocytes, referred to below as “fully grown immature oocytes.” These oocytes were mechanically freed from follicle cells and dispersed by gently shaking pieces of the ovaries with forceps into artificial seawater (ASW; Table 1). Unfortunately we were unable to obtain females close to natural ovulation from the fishery. There is as yet no method for accurately predicting the time of natural ovulation, i.e., the point at which the oocytes are naturally released from the follicle cells into the ovary’s lumen (Bumpus, 1891; Herrick, 1909). We succeeded in obtaining fully ripe females with mature ovulated fertilizable oocytes by rearing specimens in the laboratory aquaria for several weeks. In this way, we were able to obtain mature ovulated oocytes from two females, maintained in tanks supplied with running seawater, that we caught in the act of spawning. Electrophysiological Measurements Techniques for electrophysiological measurements on crab eggs have been described in detail (Goudeau and Goudeau, 1985). Briefly, experiments were performed in a 0.5-ml cell chamber sequentially perfused with artificial seawater of various composition, as listed in Table 1. The membrane potential (E,) was measured with glass fiber-filled microelectrodes (30-50 MQ in ASW when filled with 3 M KCl), inserted vertically into the oocyte. Fully grown immature and mature oocytes are coated with a tough vitelline envelope (Talbot, 1981b). This layer, which is difficult to penetrate with the microelectrode, was pierced by gently depressing the oocyte surface with the microelectrode tip and momentarily overcompensating the negative capacitance of the WPIM7 amplifier, or by giving a brief tap on the table. Ion substitution experiments were carried out to determine

VOLUME114,1986

the membrane permeability. Ionic concentration could be considerably varied by using different ASW, i.e., Cl-free, Na+-free, or K+-enriched ASW (Table 1). This procedure was useful for measuring rapidly large changes in membrane potential (AE,), but it gave only a qualitative indication of the dominant ions to which the membrane was permeable. Indeed, quantitative interpretation of ion substitution experiments requires that the change in the chemical potential of the ion be determined in solution, which is impossible with Cl-- or Na+-free ASW. The values for AE,, representing the membrane potential changes measured when normal ASW was replaced by a different ASW, were corrected for the tip potential values observed when the microelectrode was outside the oocyte (Goudeau and Goudeau, 1985). In order to measure the resistance of the egg’s plasma membrane (R,), current pulses were passed through the membrane to the external medium earthed by a 3 M KC1 agar bridge. A bridge circuit was used to pass current and to measure potential with the same electrode. In order to keep the membrane potential in the linear region of the I-V curve, +(l to 5) X lo-’ A currents were used, so that the change in membrane potential never exceeded &5 to f10 mV. Since it is difficult to measure membrane resistance accurately with one microelectrode when it is lower than 1 Ma, in these cases we only mentioned that the membrane resistance was lower than 1 MQ. Mean values are reported + the standard error of the mean, followed by n, the number of observations. In Vitro Insemination The sperm used for insemination was obtained from the thelycum or sperm receptacle of the female from which the oocytes had been collected. The thelycum is a unique ventral pouch that opens directly to the outside. Since it has no connection with the female genital ducts,

TABLE 1 COMPOSITION OFDIFFERENTARTIFICIAL SEAWATERUSED Concentration (mM)

ASW Na-free ASW K+-enriched ASW Cl-free ASW

Na+

K+

475

475

Caa+

Mg*+

cl-

12

12

20

560

12

12

20

560

487

12

20

560

12

12

20

Isethionate-

so:-

Gluconate-

Choline+

475

475

20

Tris-Cl (PH 9.1)

Tris-SO1 (PH 9.1)

(Z)

10

0

10

2.5

10

-0.5

24

a Tp corresponds to the tip potential observed when microelectrode was in the different artificial seawater (ASW).

10

-4.5

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Centrally located germinal vesicle (GV) oocytes displayed a negative resting potential of -54 + 1.5 mV (n = 32, seven females), which reached a steady maximum amplitude almost immediately after electrode penetration (Fig. la). Substitution of K+-enriched ASW for ASW led to a depolarization of the plasma membrane of 67 ? 2 mV, (n = 25, seven females) compared to 94 mV for a perfect K+ electrode. Substitution of Cl--free ASW or Na+-free choline ASW for normal ASW led to a small hyperpolarization or a depolarization of -3 +- 0.8 and 3 + 0.5 mV, respectively (n = 24, seven females). The resistance of the membrane (R,) was low, in the range of 0.8-4.5 MS2and decreased with K+-enriched ASW. These results argue against large membrane permeability to Cl- and Na+ and indicate that the central GV oocyte membrane is preferentially permeable to K+. The resting potential of the peripherally located GV oocytes (Fig. lc) averaged -40 + 1.5 mV (n = 11, three Light Microscopg Procedures females). Substitution of Cl-free ASW for ASW led to For light microscopy, some of the eggs were fixed with a membrane depolarization (Fig. lc), AE, = 8 + 2.5 mV a mixture of ethanol 95, acetic acid 50%, and formal(n = 7, three females) indicating that in these oocytes, dehyde (8/2/0.4 by volume). After fixation the vitelline the plasma membrane was slightly permeable to Cl-. coat was removed with thin needles to eliminate the Concurrently the membrane was depolarized by 53 + 4 supernumerary spermatozoa trapped in the coat. The mV (n = 11, three females) when K+-enriched ASW reeggs were then stained by a Feulgen-like reaction using placed ASW. In these peripheral GV oocytes, membrane basic fuchsin, and whole mounted. Interpretations from resistance was as low as in the central GV oocytes (range: such preparations are very precise and provide reliable 0.8-2 MQ) and when accurately measured, increased or results in the case of small-sized eggs. For larger eggs decreased with Cl-free ASW and Kf-enriched ASW, like those of lobster, this kind of investigation requires respectively. much greater technical skill and, in addition, provides In both centrally located GV and peripherally located less reliable results. Some specimens were fixed and GV oocytes (Figs. la, c), the Ca’+-H+ A 23 187 ionophore embedded in historesin (LKB) and cut into thick (5 pm) (2 X 10m6M with 12 mM Ca2+in ASW) hyperpolarized sections with glass knives on a Reichert ultramicrotome. the membrane to the same level (E,,, = -61 + 2 mV, n Other specimens were treated for electron microscopy = 6 for both types of oocytes) and decreased the memas described previously (Goudeau, 1982) and cut in semibrane resistance (results not shown). As apparent in thin (1 pm) sections. Figs. la, c, the effect of the ionophore was reversible. its opening is separated from the two female genital apertures (Herrick, 1909). The impaled mature oocytes were inseminated in vitro by bringing a small drop of sperm with fine forceps into close contact with the egg vitelline coat. During insemination, the ASW circulation into the measurement chamber was momentarily stopped. This insemination procedure is necessary because the lobster spermatozoon, like that of other decaped crustacea, lacks a flagellum and is immotile. After the electrical response to insemination occurred, the ASW circulation was resumed in the chamber. The impaled inseminated eggs were maintained in the chamber for 40 to 60 min, to allow completion of meiotic maturation, which indicated that the eggs had been activated. These eggs were then removed from the measurement chamber and processed for light microscopy.

RESULTS We first report the electrical and morphological properties of fully grown immature and unfertilized mature oocytes, because these properties provide the background for subsequent interpretation of the electrical and morphological responses of the egg to fertilization.

Mature

Oocytes

Mature oocytes were removed from the partially emptied ovaries of females in the course of spawning. This, combined with the fragility of the oocyte plasma membrane and the time required for each in vitro fertilization experiment (about 1 hr), limited these experiments to 1. MORPHOLOGICALANDELECTRICALCHARACTERISTICSabout 40 mature oocytes. In these ovulated mature oocytes, we observed that OFFULLY GROWNIMMATUREANDMATUREOOCYTES the nuclear apparatus was arrested in first meiotic metaphase (Fig. 2a). The spindle lay oblique to the Fully Grown Immature Oocytes plasma membrane, as in the crab oocyte at the same We define as “fully grown immature oocytes” all fully stage. grown oocytes that had not yet undergone their germinal In these mature oocytes, we measured a resting povesicle breakdown (GVBD). In sections, their germinal tential averaging -32 f 2 mV (range: -12 to -45 mV, n vesicles were lobed and located centrally (Fig. lb) or = 16, two females). For the measurement of membrane peripherally (Fig. Id). permeability, ion substitution experiments on mature

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b

a

t

. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . . . . . . .... . . . . .

0 -20 mV

J\ t

-40 -60

t

K=487

OCL

f

Ion0

d

mV

-6O-

, Smin , -- soap

FIG. 1. Electrical and morphological characteristics of fully grown immature oocytes of Homurus gammarus. (a) and (c) represent ionic substitution experiments and concern an oocyte with a centrally located germinal vesicle and an oocyte with a peripheral germinal vesicle, respectively. K = 487, OCL, and Iono, indicate that ASW was replaced K+-enriched ASW, Cl--free ASW or ASW + 2 X 10m6M Caz+-H+ ionophore (A 23 I87), respectively. Thin arrows pointing upwards without other indications mark the replacement of the test solution by ASW. (b) and (d) represent light micrographs of thick sections (5 pm) through an oocyte with a centrally located germinal vesicle (b), and through an oocyte with a peripheral germinal vesicle (d). Sections were stained by Shortt’s hematoxylin. (X23).

C

mV -6O-807

OCL 10 min

\flsp +\

FIG. 2. Morphological and electrical characteristics of a mature oocyte, and electrical response to insemination of H. gammas egg: (a) shows the first meiotic metaphase, in a whole-mount Feulgen preparation, of a mature fertilizable oocyte (X1615); (b) concerns an ionic substitution experiment on mature fertilizable oocyte; and (c) represents the electrical response of a mature oocyte to insemination. OCL and K = 487 indicate that the ASW was replaced by Cl--free and K+-enriched ASW, respectively. Thin arrows pointing upwards without indication response. mark the replacement of test solution by ASW. (Sp) and thick arrow indicate the insemination time. *indicates the hyperpolarization

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trinsic property of the mature oocyte’s plasma memeggs were clone as rapidly as possible after impalement, to avoid the lifting of the vitelline envelope that occurred brane. Similar results have been obtained for the mature in about 15 min, and that we detected to be ion clepen- crab oocyte (Goudeau and Goucleau, 1985). dent, as in crab eggs (Goudeau and Goudeau, 1985). Thus 2. INSEMINATIONOF THELOBSTEREGG:ELECTRICAL we tried to inseminate before this lifting occurred, since AND MORPHOLOGICAL RESPONSES OF THE EGG otherwise it might impair the sperm’s ability to reach the egg plasma membrane during the subsequent fertilization procedure. Consequently the minimal time was used to obtain a steady-state membrane potential (Figs. Electrical Response 2b, c). Ionic substitution experiments showed that at the Within 1 second after the application of a small drop mature stage, the oocyte plasma membrane is more per- of sticky sperm to the egg’s vitelline coat, we observed meable to Cl- than to other ions, as it was depolarized a rapid hyperpolarization of the egg plasma membrane by only 3 f 0.3 mv (n = 5, two females) when ASW was (400-600 ms) (Figs. 2c, 3). The rapid hyperpolarization replaced by K+-enriched ASW, but by 72 + 9 mV (n = 5, is sometimes followed by a slow hyperpolarization (from two females) when ASW was replaced by Cl--free ASW 30 seconds to several min), which brings the membrane (Fig. 2b). With normal ASW, membrane resistance was potential to a stable negative value (Figs. 3a, c, g). The low, often less than 1 MS2 (maximal value measured: 2.5 total change in membrane potential averaged -32 + 4 MQ), but it consistently increased in Cl--free ASW (5 mV (n = 9, two females). The membrane potential then 1 0.2 MSZ, n = 5). This increase in resistance argues remained steady at an average of -68 +- 0.9 mV (n = 9) against but does not eliminate the possibility of an elec- for about 1 hr, that is, the time necessary to make sure trogenic pump or an electrogenic ion-exchange mecha- that the egg reinitiated meiotic maturation indicating nism. The Ca’+-H+ ionophore A 23 187 (5 X lo-” M, with that it had been activated. The hyperpolarization usually 12 mM Ca2+in ASW) curiously had no effect on mature consisted of a one-step rise in potential (6/9 cases; Figs. oocytes (results not shown). We observed the same result 2c, 3a-e) but occasionally two-step (l/9 cases; (Fig. 3h), on mature crab eggs, which responded to the ionophore three-step (l/9 cases; Fig. 3f), or four-step rises (l/9 only when the concentration of external Ca2+was high cases; Fig. 3g) were included. (100 mM). The hyperpolarization was accompanied by a complete change in the ionic permeability of the membrane, as shown in ionic substitution experiments (Fig. 2~). The Reliability of the Electrical Measurements plasma membrane became selective for K+, as K+-enThe fully grown immature and mature oocytes of the richecl ASW and OCl- ASW depolarized and hyperpolobster are encased in a tough vitelline coat that is dif- larizecl the membrane by 83 + 5 mV and -6 rt 2 mV, ficult to pierce. The plasma membrane of the mature respectively (n = 5, two females, for both values). This oocytes appeared here to be relatively fragile, as many gain in K+ selectivity correlated with a decline in memovulated oocytes displayed a ruptured plasma membrane brane resistance: the membrane resistance of six out of with clumps of yolk in the perivitelline space. These two nine inseminated eggs that responded by hyperpolarfactors might increase the electrode impalement leak, ization was lower than 1 MS2 and could not be measured which in turn would impair the reliability of the resting after insemination. The values for membrane resistance potential measurements, However, three observations of the three remaining eggs were 5, 2.5, and 2.3 MQ, argue against such an artifact. First, fast sweep oscil- which became 1.5, 1, and 1 Ms2, respectively, following loscope records of the voltage changes that followed the the hyperpolarization response. On the other hand, in penetration of the microelectrode into the oocyte never seven unsuccessful insemination assays carried out for revealed a transient increase in membrane potential at least 40 min of impalement in ASW, the potential of (Chambers and de Armed, 1979). Second, the changes the mature oocytes remained the same. in membrane potential (AE,) in the mature oocyte when the isethionate ion, to which the membrane is imperme- Morphological Changes in the Egg after Insemination able, replaced chloride sometimes reached values greater and the Electrical Response than 90 mV. These values were considerably higher than The impaled eggs that responded to insemination by those calculated for a trivial diffusion potential at the hyperpolarization were maintained in the perfusion site of a membrane leak (Jaffe et aL, 1979). Third, the chamber for 40 to 60 min. They were then removed and membrane resistance for the mature oocyte, which was observed by light microscopy after appropriate treatin the range of
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FIG. 3. Oscilloscope records of fertilization potentials in H. gammorua For all figures, the upper, middle, and lower traces represent respectively the 0 potential level, one or several sweeps at the resting potential level, and the membrane potential after the hyperpolarization response. Note that in (f), (g) and (h), the hyperpolarization response proceeds in several steps, and that in most of the experiments a secondary, slow hyperpolarization occurs. Vertical and horizontal bars correspond to 20 mV and 2 seconds, respectively, for all the components of the figures.

GOUDEAU

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GOUDEAU

nated eggs exhibited two distinct morphological tures:

fea-

1. In four out of nine eggs inseminated in vitro, we observed formation of the first polar body 1 hr after hyperpolarization: for two eggs the first polar body was observed in semi-thin sections (Figs. 4, 5), and for the

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two others in whole-mount preparations where we were able to detect the second meiotic metaphase by staining with Feulgen’s reaction (Fig. 9). At the periphery of the polar body, we observed in the semi-thin sections that the plasma membrane was deeply infolded (Fig. 4). This localized activation of the cortex has also been observed in crab eggs (unpublished results). In whole-mount

FIGS. 4-9. Light micrographs of fertilized eggs of H. gammarus fixed 1 hr after insemination and the concurrent hyperpolarization response. These eggs were in the process of resuming meiotic maturation. Figures 4 and 5, from semi-thin sections (1 pm) stained with toluidine blue, show complete first polar bodies in the eggs that displayed the hyperpolarization response depicted in Figs. 3a and b. Note the highly indented plasma membrane in the polar body area of Fig. 4, and the residual chromosomes (ch) in the polar body of Fig. 5; vit c, vitelline coat; y, yolk globule. (X1400). Figures 6,7, and 8 show three serial semi-thin sections (1 pm), stained with toluidine blue, through the cortical region of the egg that developed the hyperpolarization response shown in Fig. 3a. Notice the spermatozoon nucleus completely included in the cortex (6). There is no sperm component inside or outside the vitelline coat (vit c). In sections shown in Figs. 7 and 8, a rod (R) can be observed, located in front of the penetrated nucleus (X1400). Figure 9 shows the second meiotic metaphase spindle (sple) detected in a whole-mount Feulgen preparation of the fertilized egg that displayed the hyperpolarization response shown in Fig. 3c (X1200).

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preparations of three eggs that were unsuccessfully in- Maturation-Related Changes in Oocyte Membrane seminated and that did not show a hyperpolarization Permeability response, we observed first meiotic metaphase but no polar body formation. The membrane of the fully grown immature lobster 2. Of seven inseminated eggs that displayed hyper- oocyte is K+ selective, as in crabs and other animal polarization, and for which membrane potential was groups (Goudeau and Goudeau, 1985;review in Hagiwara and Jaffe, 1979). During maturation, K+ selectivity is monitored for 1 hr, only two showed in whole-mount lost but we did not detect any concomitant increase in preparations Feulgen-positive structures which obviously were sperm nuclei. However, we considered the membrane resistance, as was observed in crab oocytes whole-mount method difficult to handle, since the sperm (Goudeau and Goudeau, 1985). nucleus is about 9 pm in width and the egg about 1.8 In the mature lobster oocyte, the plasma membrane mm in diameter. Under these unfavorable conditions of is predominantly permeable to Cl-, as in crabs; this difinvestigation, it is very likely that we failed to detect fers from the permeability reported for other animal sperm penetrating into the five other eggs processed for groups (Goudeau and Goudeau, 1985; Hagiwara and whole-mount observation. So we analyzed two insemi- Jaffe, 1979). This plasma membrane permeability to nated eggs, in semi-thin sections of specimens fixed and chloride, established for both macruran (H. gammarus) embedded in resin. This method of investigation ap- and brachyuran (Car&us maenas and Maia squinado) species, may reflect a general property of mature cruspeared to be more reliable even if more time consuming than whole-mount preparations. In serial sections of two tacean oocytes. Further results for anomurans (e.g., the eggs, we could detect both the first polar body (Fig. 4) hermit crab) and natantians (e.g., shrimps and prawns) and the fertilizing spermatozoon nucleus which had might confirm this hypothesis. penetrated into the egg cortex (Figs. 6-8). In addition, we clearly observed in serial sections other sperm com- Nature of the Electrical Response to Insemination ponents, such as a rod located in front of the nucleus and linked to it by a narrow, faintly contrasted structure Insemination triggered a rapid and sustained hyper(Figs. 7,8). This rod might correspond to the acrosomal polarization of the egg plasma membrane caused by infilament, according to the scheme of the reacted lobster creased membrane permeability to K+. We have not yet spermatozoon proposed by Talbot and Chanmanon identified the mechanism that promotes the sudden in(1980). crease in membrane permeability to K+, but we tentatively propose that a rise in cytoplasmic-free Ca2+, of To conclude, these morphological observations proved either intra- and/or extracellular origin, might mediate the hyperpolarization, since in other species fertilization that the inseminated eggs displaying membrane hypertriggers a dramatic rise in (Ca2+)i (review in Jaffe, 1983). polarization were fertilized. This increase in (Ca2’)i might promote a Ca2+-activated K+ conductance (Meech, 1978), as reported for the hamDISCUSSION ster egg (Igusa and Miyazaki, 1983). In support of this rise in (Ca2+)i, we observed that application of the Ca2+In this paper we analyzed the electrophysiological H+ ionophore A 23 187 (2 X lop6 M in ASW with 12 mM properties of fully grown immature and mature lobster Ca2+) to fully grown immature lobster oocytes caused oocytes, and we established the electrical and morphomembrane hyperpolarization. Curiously the ionophore logical responses of the egg to insemination. Our major had no effect on mature oocytes. This observation refindings are: (i) In the mature oocyte, the plasma mem- quires further investigation. brane, which previously was predominantly permeable to K+, is mainly permeable to Cl-. (ii) The electrical rePotential of the Lobster Egg sponse of the egg to insemination consists of a fast sus- The Fertilization tained hyperpolarization of the plasma membrane. (iii) Several arguments lead us to conclude that the egg This response is correlated with the resumption of membrane hyperpolarization response to insemination meiosis. A sperm nucleus was observed in the cortex of corresponds to the fertilization potential of the lobthese eggs which was taken as proof that inseminated egg displaying a hyperpolarization response had been ster egg: fertilized. Accordingly, the hyperpolarization response (i) Mature unfertilized oocytes have a nuclear appamight be considered as the fertilization potential of the ratus arrested at first meiotic metaphase. By 1 hr after lobster egg. This will be discussed below.

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fertilization (Talbot and Chanmanon, 1980). This reacin vitro insemination, and the concurrent hyperpolarization response, the first polar body had formed in four tion was induced artificially by the Ca2+-H+ A 23 187 of the nine eggs tested. From semi-thin section obser- ionophore and appeared to be extremely fast (about 1 second; Talbot and Chanmanon, 1980). Up to the present vations, the plasma membrane appeared deeply infolded time, no data have appeared about the time course of in the vicinity of the polar body. These morphological the acrosome reaction and the concurrent gamete conresponses indicated that these eggs had been activated. (ii) The nucleus of the fertilizing spermatozoon was tacts occurring during in viva fertilization in the lobster. present in the cortex of three of these activated eggs Our present electrophysiological study indicates that the that displayed the hyperpolarization response. This egg electrical response happens practically instantacomplementary result provides evidence that these eggs neously when the sperm is brought into close contact with the egg coat. We were not able to measure accuwere fertilized. That cleavages could be correlated with the occurrence of the fertilization potential would con- rately the duration of this physiological acrosome revincingly strengthen this assertion. Unfortunately we action, yet we estimated it to last less than 1 second. In view of the Talbot’s observations, our present data apnever could keep crustacean eggs, assumed to be fertilized, alive for more than 12 hr after removal of the mi- pear to be sound and lead us to assume that the hypercroelectrode. Indeed, this is the time necessary to obtain polarization response is correlated with gamete contact two-cell stages from the yolky crab eggs, under our in- at fertilization. (v) Finally, our results about in vitro fertilization in cubation conditions, following bulk in vitro insemination crab eggs, founded on numerous experiments, might without previous impalement. (iii) Seven oocytes that were not fertilized did not support our present results on lobster fertilization, since there is a striking similarity of the egg electrical redisplay any change in membrane potential, thus providing evidence that neither the impalement procedure, nor sponse to insemination in these two models. In crabs, the effect of ions in ASW was involved in triggering the we found that 60% of the inseminated eggs that dishyperpolarization. In addition, morphological observa- played a hyperpolarization response (n = 46) were fertions of whole mounts done on six of these eggs and the tilized, since they resumed their meiotic maturation and histological analysis of one, permit us to confirm that contained without exception, fertilizing spermatozoon (unpublished data). these eggs had not been activated and did not contain any penetrating spermatozoon. Furthermore, we would In conclusion, we propose that the egg membrane hystress that during the normal insemination procedure, perpolarization corresponds to the fertilization potential that is when the ASW circulation was momentarily stopped in the measurement chamber, not all the fer- of the lobster egg. tilization attempts were successful. This experimental condition impairs the dilution of the sperm solution and, Physiological Significance of the Hyperpolarixing consequently, would favor the activation by some nonFertilization Potential sperm constituent. In fact, that no electrical and morphological response occurred under these conditions, The electrical and morphological responses to fertilrules out the probability of action of a nonsperm con- ization are strikingly similar in lobster and crab eggs. stituent. Therefore, whether fertilization occurs internally in the (iv) The unusual pattern of acrosome reaction of the female genital duct (crab), or externally in seawater lobster immotile spermatozoon may explain why the egg (lobster) apparently does not affect the egg’s responses electrical response occurred so soon after insemination to fertilization. and concurrent fertilization; thus, the possibility that The electrical response of crab and lobster eggs to the hyperpolarization response might not be related to fertilization consists of a rapid and sustained hyperthe fertilization process can be eliminated. Indeed, the polarization caused by an increase in Kf conductance of lobster spermatozoon follows the general reptantia pat- the egg membrane. As this gain of Kf conductance durtern of sperm reaction (review in Talbot and Chanma- ing fertilization was reported for sea urchins, echiurians non, 1980) that can be summed up as resulting from the (Hagiwara and Jaffe, 1979), amphibians (Jaffe and eversion of the spermatozoon, i.e. the spermatozoon ap- Schlichter, 1985), fish (Nuccitelli, 1980), hamster (Igusa pears to be turned inside out. This reaction, which is and Miyazaki, 1983;Igusa et al, 1983), and, more recently, concomitant with sperm penetration of the oocytic en- in the nemertean worm Cerebratulus lacteus (Kline et velopes, generates a slight forward movement of several ak, 1985), it seems to be a general ionic mechanism resperm components and permits the otherwise immotile lated to fertilization. However, it is clear that there is sperm to ranidlv attain the egg nlasma membrane at a large variability in the onset of this K+ conductance - ” YU A

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increase. Indeed, in sea urchin, echiurian, fish, amphibian, and nemertean eggs, increase of K+ conductance follows an early sperm-triggered increase in membrane conductance to Na+ or Cl-, of variable duration, that first depolarizes the egg membrane and might mask some early K+ conductance, as demonstrated in amphibians (Jaffe and Schlichter, 1985). By contrast, in the hamster egg, the increase of K+ conductance occurs without any previous depolarization of the egg membrane and promotes transient hyperpolarizations superimposed to a slow hyperpolarization of the membrane, several seconds after gamete contact. Finally, in crab and lobster eggs, this increase of K+ conductance arises without any early membrane depolarization, thus is similar to what has been reported for hamster eggs. But, in the two crustacean models, K+ conductance increases within about 1 second after insemination. In fact, this increase appears to require only about 500 ms, and it is sustained. Both the absence of a depolarizing step and the fact that hyperpolarization is sustained are intriguing features of the fertilization potential, in lobster eggs, whose physiological significance might be hypothetized. In this connection, the initial depolarization observed in response to fertilization in the eggs of sea urchins, nemertean worm, echiurians, and amphibians, provides a fast and transient electrical block to polyspermy at the plasma membrane level (Jaffe, 1976; Nuccitelli and Grey, 1984; review in Gould-Somero and Jaffe, 1984). The inverted electrical response of the lobster egg might also provide an electrical block to polyspermy. Yet, this egg might have additionally a nonelectrical block at the vitelline coat or plasma membrane or might be physiologically polyspermic. In crabs, our data showed that 50% of eggs fertilized in vitro exhibited polyspermy, since two to four spermatozoan nuclei entered 38% of the eggs and 10 to 12 spermatozoon nuclei penetrated 12% of the eggs (n = 38). The number of nuclei correlated relatively well with the number of steps observed in the hyperpolarization response (unpublished results). Curiously, this multistep electrical response appeared to be less frequent in lobster than in crab eggs, but we have as yet no statistical data for the lobster. Whether decapod crustacean eggs are physiologically polyspermic remains open to question. We thank Laurinda Jaffe for her helpful comments, and Mr. Cabioch of the fishery “La Langouste” who helped us obtain the appropriate lobsters. We are grateful to Annick Dorme and Franpoise Kleinbauer for their helpful technical assistance. We also thank our friend Mathilde Dreyfus for stylistic improvement of the manuscript. This work was supported by grants from IFREMER, CNRS and “Aide a la recherche universitaire.”

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