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Absence of a complete block to polyspermy after fertilization of Mytilus galloprovincialis (Mollusca, Pelecypoda) oocytes
DEVELOPMENTAL
BIOLOGY
97,27-33
(1983)
Absence of a Complete Block to Polyspermy after Fertilization of /WyU/us galloprovincialis (Mollusca, Pelecypoda) Oocytes LOUISE Dummm-DuBk,**t “Station
FRANCOIS DuBb,**t PIERRE GUERRIER,*~’ AND PIERRE CouILLmDj-
Biologique, 29211 Roscoff, France; and TDkpartement des Sciences Biologiques, Universiti de Mont&al, C P. 6128, Mont&al, Q&bec, H3C 3J7 Canada Received July 1, 19X2; accepted in revised form December 1.3, 1982
Mytilus galloprovincialis oocytes undergo monospermic fertilizations (1 sperm nucleus/oocyte) over a wide range of sperm-oocyte ratios beyond which the number of penetrating sperm increases either linearly or exponentially over 10 min. Artificial activation of oocytes by KC1 or the ionophore A 23187, up to the polar body extrusion stage, allows successful fertilizations upon a subsequent insemination. No organized and complete detachment of supernumerary oocyte-bound sperm is detected after fertilization. Reducing the external Na+ concentration promotes a higher rate of fertilizations. These results suggest that no complete block to polyspermy is established in this species but that a partial block, Na+ dependent, might be sufficient to ensure monospermic fertilizations under natural conditions. INTRODUCTION
A successful fertilization results in the fusion of one male pronucleus to a female pronucleus and the further development of the zygote may then proceed normally. In species for which the penetration of more than one spermatozoon is the rule (physiological polyspermy), for example, pulmonate molluscs (Raven, 1966), urodeles (Fankhauser, 1948), elasmobranchs (Wourms, 1977), reptiles (Rothschild, 1956), and birds (Romanoff, 1960), monospermy is ensured at the level of the egg cytoplasm (for a review, see Austin, 1965). In most animal species, however, when more than one spermatozoon penetrate in the egg, the resulting polyspermy leads to the death of the embryo (Austin, 1965). To prevent the lethal effect of polyspermy, it is generally agreed that a specific defense mechanism, the so-called “block to polyspermy,” is set up by the egg to allow only one sperm to penetrate. In the well-studied sea urchin egg, the block to polyspermy appears to proceed in two distinct steps. First, a rapid partial block lowers the receptivity of the egg plasma membrane to further sperm fusions in less than 2-3 set after the first fertilization (Rothschild and Swann, 1952), this block being electrically mediated (Jaffe, 1976). One to two minutes after, this partial block is followed by a cortical granule exocytosis and the elevation of a fertilization coat around the egg which establishes a complete, but slow, block to polyspermy. However, the existence of a rapid partial block to polyspermy preceding the onset of the cortical reaction in sea urchin eggs has been and still is questioned by many 1 To whom reprint
requests should be addressed.
workers (Hagstriim, 1956; Byrd and Collins, 1975; De Felice and Dale, 1979;for a review, see Dale and Monroy, 1981). In the oocytes of the echiuran Urechis, a fast block to polyspermy begins to develop in less than 10 set after insemination (Paul, 1975a). Even though, in this species, surface coat reorganization occurs 4 min after fertilization, it has probably no role in polyspermy prevention (Paul and Gould-Somero, 1976). It has been later demonstrated that the fast partial block is directly related to a transient oocyte membrane depolarization, lasting around 10 min after fertilization (Gould-Somero et al., 1979). This is followed by a complete block of unknown nature at the level of sperm-egg plasma membrane fusion (Paul and Gould-Somero, 1976). In pelecypod mollusc oocytes, the cortical granules, though present, do not undergo exocytosis nor does any extracellular coat elevate upon fertilization (Pasteels and De Harven, 1962; Rebhun, 1962; Humphreys, 1967). Nevertheless, a complete block to polyspermy has been shown to take place in less than 15 set after fertilization (Ziomek and Epel, 1975) or after artificial activation (Longo, 1976) of Spisula oocytes. Our paper extends these earlier investigations to a second pelecypod mollusc, Mytilus galloprovincialis, and presents evidence that, contrary to Spisula, no complete block to polyspermy is established in the few minutes following fertilization or artificial activation of Mytilus oocytes. This result raises the question of the maintenance of monospermic fertilizations under natural conditions. A working hypothesis is presented to account for this original finding, an absence of complete block, and is also proposed as a suitable model to reconcile
27 0012-1606/83 $3.00 Copyright All rights
0 1983 by Academic Press, Inc. of reproduction in any form reserved.
28
DEVELOPMENTAL BIOLOGY
conflicting results on the fast block to polyspermy in sea urchin eggs. MATERIALS
AND
METHODS
Mytilus gallwpmvincialis were collected at Concarneau (Brittany) and kept in running seawater at the Station Biologique de Roscoff. Spawning was induced by placing the mussels in natural filtered seawater (FSW) at 30°C for 30 min, then in FSW at 4°C for 30 min. Spawning generally starts after returning the animals in FSW at room temperature. The sperm was collected “dry” and kept at 4°C until use. Working sperm suspensions were prepared by diluting the initial stock with FSW or artificial seawater (ASW) and sperm concentrations were determined turbidometrically at 680 nm, according to the equation: Y (sperm/ml) = 8.98 X OD X 10’. This equation was determined according to Rothschild (1950), by counting with a hemocytometer the number of sperm per milliliters in suspensions of various optical densities. The oocytes (naturally blocked at first metaphase of maturation) were washed several times with either FSW, ASW, or sodium-free seawater (NaFSW) acording to the experiments, and were kept at 15°C until use. Oocyte concentrations were determined by counting the number of oocytes in lo-p1 glass capillaries, taken from the working suspension. ASW was prepared according to the MBL formula (Cavanaugh, 19’75),with the addition of 2 mMTris (hydroxymethyl)-aminomethane. NaFSW had the same formula as ASW, except that NaCl was replaced by the molar equivalent of choline chloride. The pH was adjusted to 8.1-8.2 with NaOH for ASW and with KOH for NaFSW. Low-sodium seawater (LowNaSW) of variable final Na+ concentrations was prepared by mixing proper amounts of NaFSW and ASW. The incidence and severity of polyspermy were determined by counting the number of decondensed sperm nuclei in oocytes fixed 20 to 30 min after insemination in a 3:l mixture of ethanol:acetic acid for 1 hr, cleared in 55% acetic acid for 30 min, and kept in 70% ethanol (adapted from Byrd and Collins, 1975). Observations were done under phase-contrast microscopy, by counting 50-100 oocytes per sample. The time course of fertilizations was assayed by adding 0.001% to 0.01% sodium lauryl sulfate (SLS) to the gametes at various times after insemination (Hagstrom and Hagstriim, 1954). SLS inactivates all unbound sperm except those already fused with the oocyte plasma membrane (Byrd and Collins, 1975). In the case of Mytilus, this was tested by incubating sperm in different concentrations of SLS for 10 set, then adding these sperm to oocytes. No fertilization occurred above 0.0005% SLS;
VOLUME 97, 1983
to insure that at high-sperm concentrations, all unbound sperm were inactivated, higher concentrations were used, the eggs being washed 10 set after addition of the detergent. The time course of sperm-oocyte binding was determined by the subtraction method of Vacquier and Payne (1973), by inseminating a single batch of oocytes and by fixing aliquots at desired times in 1% glutaraldehyde in ASW. Artificial activation was achieved by adding either 10 PALM A 23187 (from a stock solution at 5 mM in 100% ethanol) or 20% v/v isotonic KC1 (0.52 M) to the oocyte suspensions for 1 hr. The percentage of activated oocytes was determined by counting the number of oocytes having extruded their polar bodies. The suspensions were then washed four times and inseminated. The percentage of fertilized oocytes was determined by counting the number of cleaved oocytes, 2 hr after insemination. All experiments were carried out at room temperature (18°C). Additional details are given in the text or figure legends. RESULTS
The relationship between the number of sperm penetrations and the sperm-oocyte ratio (Fig. 1) reveals that successful fertilizations (e.g., 1 sperm/oocyte) are obtained over a range of sperm-oocyte ratios between 10’ to 2 X 10’ (Table 1). Beyond these sperm-oocyte ratios, there is, at first, a monotonic increase in polyspermy (between 10zto lo3 sperm/oocyte) while over l5 X 103, the number of penetrating spermatozoa increases at an exponential rate. The sperm-oocyte ratios at which the change from monotonic to exponential increase occurs is very narrow (Fig. 5), and may vary, as mentioned above (l-5 X 103)from batch to batch. These observations are detailed on Table 1, where one can see that for different sperm densities, polyspermy increases in harmony with increasing sperm-oocyte ratios. Thus, monospermy is the rule only when the sperm-oocyte ratio is low, whatever the sperm density is. The number of penetrated spermatozoa for different sperm-oocyte interaction times, and ratios, was determined following the method of Byrd and Collins (1975). Examples of the three types of curve currently obtained are presented on Fig. 2. At low sperm-oocyte ratios, the first fertilization takes longer to be effected (it can be as long as 10 min) but the number of penetrating spermatozoa levels off at around 1 sperm/egg (Fig. 2A). When sperm-oocyte ratios are increased, the number of penetrating spermatozoa first tends to be in a linear relation with time (Fig. 2B) and, at higher ratios, tends to rise exponentially (Fig. 2C). These results demonstrate that over the time period tested (10 min), no complete block to polyspermy is established.
Fertilizatim
DUFRESNE-DUBS ET AL.
of Mgtilus
103
102
Sperm-oocyte
29
galloprovincialis
10
ratio
FE. 1. Induction of polyspermy by increasing sperm-oocyte ratios. Eighteen samples, 1 ml each, of ooeytes from one female, ranging from 4 X lo3 to 9 x 10” oocytes/ml, were inseminated with four different sperm densities, ranging from 1.86 X 10” to 9.38 X 10’ sperm/ml. Two minutes after mixing the gametes, the oocytes are washed and fixed 20 min after insemination. Sperm nuclei were counted in 50-100 randomly selected oocytes for each sample.
This is further suggested by the fact that oocytes artificially activated by KC1 or the calcium ionophore A 23187 and having extruded their polar bodies, are fertilizable upon subsequent insemination (Table 2). Observations of other KCl-treated oocytes revealed that TABLE 1 INDUCTION OF PQLYSPERMY BY INCREASING SPERM-O• CYTE RATIOS, FOR DIFFERENT SPERM DENSITIES Sperm Densities (sperm/ml) 1.87 x 106
1.87 X lo7
6.25 X lo7
9.38 x lo7
Sperm/oocyte
ratios
2.06 2.78 4.61 8.33 2.08
X X X x X
2.05 3.05 4.54 9.07 2.26
x lo*
1.03 1.37 2.06 4.12 1.03
10’ 10’ 10’ 10’ 102
x lo2 x 102
x 10’ X IO3
x lo3 x 103 X IO3 x lo3
x lo4
2.06 X lo3 2.68 X lo3
4.12 X lo3
Mean number of sperm/oocyte 1.05
1.08 1.34 1.43 1.53 2.06 2.31 3.67 4.62 19.46 3.55 4.62 6.98 13.06 50.10 3.67
13.98 21.10
more than 80% of oocytes contain at least one sperm pronucleus after insemination. This observation provides evidence that cleavages reported in Table 2 involved sperm penetrations and were not mere “parthenogenetic activation” without sperm fusion and incorporation. The extrusion of polar bodies normally takes place 20-25 min after fertilization at 20°C. Sperm-oocyte binding assays (Fig. 3) reveal that sperm bind mostly during the first 15 see after insemination, but that no full detachment will occur over the following 10 min. The effect of lowering the external Na+ concentration for different sperm-oocyte ratios (Fig. 4) shows that, for low ratios, a 20 mM external Na+ concentration, or below, induces a small increase of polyspermy (mean number of sperm/oocyte). However, higher sperm-oocyte ratios, only slightly polyspermic at normal Na+ concentrations (425 mM), induce a four- to fivefold increase in the number of penetrated sperm when the external Nat concentration is below 300 mM. These results suggest the possibility of a cumulative negative effect on the oocyte defense mechanism when high sperm-oocyte ratios and low-sodium seawater are used at the same time. DISCUSSION
Even though polyspermic fertilization of M&ilus oocytes leads to abnormal cleavages and eventually to the death of the embryo, our results demonstrate that no
30
DEVELOPMENTAL BIOLOGY
VOLUME 97, 1983 TABLE 2 FERTILIZATION OF ARTIFICIALLY ACTIVATED Mytilus
OOCYTES
12-
Treatment
Percentage of oocytes having extruded first polar body after 90 min”
Percentage cleavages of treated oocytes 120 min after inseminatior?
A23187 (10 @f) KC1 (20%, v/v)
96.3 95.5
90.2 98.8
11 -
lo-
0
I
Note. Mean results of two experiments with two batches of oocytes. a None of the unstimulated control oocytes or oocytes submitted to 0.2% ethanol extruded polar bodies. * None of the KC1 or A23187-activated oocytes cleaved without insemination. Additional experimental procedures are given under Materials and Methods.
2-
I
Time
t
1
I
I
I
I
I
n
Spi.sula oocytes are at the germinal vesicle stage (prophase I). This difference in the physiological conditions of Spisula and Mytilus oocytes has been related to the fact that Spi.sula oocytes release acid upon fertilization (Guerrier et al., 1981) while Mytilws oocytes do not (Paul, 19’7513; Dub6 and Guerrier, 1982). The present paper adds new results to the growing list of physiological differences between Spisula and Mytilus oocytes. How can an animal species resist the lethal effect of polyspermy without an absolutely efficient defense mechanism? In an attempt to answer this question and according to our results, a schematic working hypothesis is presented (Fig. 5). Under reasonable sperm-oo-
5 6 7 8 9 10 of transfer in SLS (min.)
FIG. 2. Severity of polyspermy for different sperm-oocyte interaction times. A single batch of oocytes is inseminated at 0 min. At the indicated times, oocytes are transferred into 0.901% SLS (A) or 0.01% SLS (B and C) for 3 min, washed, and fixed 20 min after insemination. Final sperm-oocyte ratios: (A) 1.2 X 10’; (B) 3.0 X 103; (C) 4.4 x los.
specific defense mechanism completely protects the oocyte from this “deleterious” possibility. This result is consistent with the fact that, upon fertilization of i@tilus oocytes, no extracellular coat elevates which, in turn, would form an absolute barrier to further sperm penetrations as is the case for sea urchin eggs. However, our results contrast those obtained for Spi&a oocytes which establish a complete block to polyspermy in less than 15 set after the first fertilization (Ziomek and Epel, 1975). Moreover it has also been shown that Spisula oocytes artificially activated by a treatment with hypertonic seawater (Longo, 1975) cannot be subsequently fertilized. This differs from our results on KC1 and A23187 activations (Table 2). It must be kept in mind that, at the time of fertilization, M@ilus oocytes are arrested at the metaphase I of meiotic maturation while
i 800-
o
1
2 Time
3 4 5 of transfer
6 7 8 9 IO in glutaraldehyde(min)
FIG. 3. Time course of sperm-oocyte binding. A single batch of oocytes is inseminated with the same volume of a sperm suspension. At the indicated times, 2 ml of gametes is transferred into 2 ml of 2% glutaraldehyde in seawater. The optical density of the supernatant is then measured. The number of bound spermatozoa is obtained by calculating the difference between the total number of spermatozoa available and the number of unbound sperm. It is then divided by the number of oocytes per milliliter. Final sperm-oocyte ratios: (0) 3.97 x 10s; (0) 4.95 x lo?
Fertilization of
DUFRESNE-DUBS ET AL.
mc z
. O
n
n 100
’
’ 200
.
1 300
Na+Concentration
. 400
(mM)
FIG. 4. Induction of polyspermy at different centrations. Oocytes were incubated in 10 ml of NaSW for 15 min, and inseminated. Interaction for 20 min, and then the oocytes were fixed. Final (A) 1.69 X 10’; (B) 9.89 X lo*; (C) 1.77 X 103.
external sodium conASW, NaFSW, or low of gametes occurred sperm-oocyte ratios:
cyte ratios, monospermy is the rule while excessive sperm-oocyte ratios result in “explosive” polyspermy. This could imply that a partial block to polyspermy ensures monospermic fertilizations under sperm-oocyte
OQCYTE DEFENCE AGAINST POLYSPERMY
Mytilus
galloprcn+xialis
ratios likely to reflect the situation prevailing under natural conditions. Under experimentally imposed conditions (excessive sperm-oocyte ratios), the efficiency of this partial block to polyspermy rapidly decreases down to a level at which it becomes completely overwhelmed. A decrease in egg receptivity to spermatozoa as soon as the first fertilization is effected would lower the probability of a successful1 second fertilization. This hypothesis was first proposed by Rothschild and Swann (1952) for the sea urchin egg. If one assumes that this first partial block is related to a transient membrane depolarization (Jaffe, 1976) it appears likely that the efficiency of this partial block would fluctuate with time since such an initial membrane depolarization is followed up by a gradual hyperpolarization in all the marine invertebrate eggs so far studied (Hagiwara and Jaffe, 1979). The relative efficiency decrease of the oocyte defense mechanism may also be related to the cumulative effect of extruded sperm lysins digesting the oocyte extracellular coats which appear likely to play a determinant role in the protection against polyspermy (Hagstrom, 1956; Dale and Monroy, 1981). On the other hand, an increase of the sperm concentration is also likely to result in a higher “collision rate” between sperm and oocytes.
FERTILIZING ACTIVITY
SPERM-OOCYTERATIO
J2 $8 "6
a
? 1
ATTAIN A PLATEAU AT 1 SPERM PER OOCYTE
LINEAR
(NATURAL CONDITIONS ?) FIG. 5. Schematic
working
hypothesis
31
EXPONENTIAL
(LABORATORY IMPOSED CONDITIONS ?) on the block to polyspermy
in Mytilus
oocytes.
32
DEVELOPMENTAL BIOLOGY
Such an antagonistic effect of raising sperm-oocyte ratios on the oocyte defense mechanism and on the sperm fertilizing activity (Fig. 5) would explain the rapid increase of fertilization rates depicted in Figs. 1 and 2. In ascidian eggs which also lack a cortical reaction, Lambert and Lambert (1981) have shown, by the method of a first monospermic insemination, followed at various time intervals by a second polyspermic insemination, that these eggs could be incapable of refertilization by a second spermatozoon about 21 see after the first fertilization. However, Lambert and Lambert (1981) further mentioned that the half-conduction time for the establishment of this block to polyspermy could require more than 5 min when higher sperm concentrations were used. With Myths oocytes, we have also obtained, by the same method, highly variable estimations of the conduction time of the establishment of the block to polyspermy, ranging from 2 min to the complete absence of any block to polyspermy by 10 min, depending on the sperm-oocyte ratios used (data not shown). Lambert and Lambert (1981) claimed that the timing for the establishment of the block to polyspermy in ascidian eggs is dependent upon the sperm concentration. This seems to us the indication of an incomplete partial block fitting well with our proposed model (Fig. 5). The involvement of extracellular Naf in the maintenance of a lower fertilization rate (Fig. 4) supports the hypothesis that Na+-dependent membrane depolarization is related to a partial block of Mytilus oocytes as is the case in Ascidians (Lambert and Lambert, 1981) and in Urechis for which it has been demonstrated that low-sodium seawater reduced the fertilization potential level, thus allowing supernumerary spermatozoa to penetrate (Gould-Somero et al., 1979). Whatever the exact nature of this block is, we suspect a possible similarity with the presumed fast block to polyspermy of sea urchin eggs. Measurements of fertilization rates in sea urchin eggs, determined by counting sperm nuclei, reveal either a rate of second sperm penetration lower than the rate of first sperm penetration (Presley and Baker, 1970) or a linear rate of sperm penetrations strictly dependent upon the sperm-egg ratio (Byrd and Collins, 1975). A careful examination of their data may lead one to interpret the rate of sperm penetrations, at the highest sperm-egg ratio tested, as being exponential rather than linear (Fig. 3A of Byrd and Collins (1975)). These three types of observations roughly correspond to our results (Fig. 2) which stress the fact that fast partial blocks to polyspermy might not be processes resulting in a fixed decrease of the egg membrane receptivity to spermatozoa. Therefore, previous determinations of sea urchin egg fertilization rates might not be contradictory as would seem at first sight, but
VOLUME 97, 1983
would reflect a real, though complex, situation. The main problem in studying the fast partial block to polyspermy in sea urchin eggs is that it is experimentally difficult to separate its possible role from the obvious one of the cortical reaction which may begin as soon as 20 set after the first fertilization. To understand better the nature of these partial blocks to polyspermy, Mytilus oocytes might therefore provide a new, promising material for investigation. Also promising would be investigations on the precise nature of the complete blocks to polyspermy in Urechis (Paul and Gould-Somero, 1976) and Spisula oocytes (Ziomek and Epel, 1975). Mrs. Claude Guerrier is acknowledged for drawing the figures and Mrs. Nicole Guyard for typing the manuscript. We also deeply thank Drs. Michael J. Whitaker and Brian Dale for valuable comments on the first draft of this paper. Thanks are due to an anonymous reviewer for helpful suggestions and requests. This work was supported by FCAC (Quebec) Ph.D. scholarships to L.D.D. and F.D., by a DGRST (France) grant (ACC 79.70777) to P.G. and a NSERC (Canada) grant to P.C. REFERENCES Prentice-Hall. Englewood Cliffs, AUSTIN, C. R. (1965). “Fertilization.” New Jersey. BYRD, E. W., and COLLINS,F. D. (1975). Absence of fast block to polyspermy in eggs of sea urchin Strungylocentrotus purpuratus. Nature (London) 257, 675-677. CAVANAUGH,G. M. ed. (1975). “Formulae and Methods VI of the Marine Biological Laboratory.” Woods Hole, Mass. DALE, B., and MONROY,A. (1981). How is polyspermy prevented? Gamete Res. 4, 151-169. DE FELICE, L. J., and DALE, B. (1979). Voltage response to fertilization and polyspermy in sea urchin eggs and oocytes. LIev. Biol. 72,327341. DUB& F., and GUERRIER, P. (1982). Acid release during activation of Barnes can&i& (Mollusca, Pelecypoda) oocytes. Deu. Growth &fferen. 24.163-171. FANKHALJSER, G. (1948). The organization of the amphibian egg during fertilization and cleavage. Ann N. Y. Awd Sci. 82.684-708. GUERRIER, P., DUB& F., and MOREAU, M. (1981). External calcium requirements for oocyte maturation in the surf clam Spisula solidissimu Biol Bull. 161, 335-336. GOULD-SOMERO, M., JAFFE, L. A., and HOLLAND, L. (1979). Electrically mediated fast block in eggs of the marine worm Urechis ~aupu..J.
Cell BioL 82.426-440. HAGIWARA, S., and JAFFE, L. A. (1979). Electrical properties of egg cell membranes. Annu. Rev. Biophys. Bioeng. 8,385-416. HAGSTR~M,B. E. (1956). Studies on polyspermy in sea urchins. Arkiv. ZOOL 10,307-315. HAGSTR(JM, B., and HAGSTROM,B. (1954). The fertilization rate in sea urchins. Exp. Cell Res. 6,479-484. HUMPHREYS,W. J. (1967). The fine structure of cortical granules in eggs and gastrulae of Mytilus edulis J. Ultra&-u&. Res. 17, 314326. JAFFE, L. A. (1976). Fast block to polyspermy in sea urchin eggs is electrically mediated. Nature (London) 261, 68-71. LAMBERT,C. C., and LAMBERT, G. (1981). Formation of the block to polyspermy in ascidian eggs: Time course, ion requirements, and role of the accessory cells. J. Exp. Zool. 217, 291-295. LONGO, F. J. (1976). Ultrastructural aspects of fertilization in spiralian eggs. Amer. Zoo1 16,275-394.
DUFRESNE-DUBI? ET AL.
Fertilization
PASTEELS, J. J., and DE HARVEN, E. (1962). Etude au microscope electronique du cortex de l’oeuf de Barnea candidu (Mollusque bivalve), et son evolution au moment de la fecondation, de la maturation, et de la segmentation. Arch Biol. (Liege) 73,465-490. PAUL, M. (1975a). The polyspermy block in eggs of Urechis caupo. Evidence for a “rapid” block. Exp. Cell Res. 90.137-142. PAUL, M. (1975b). Release of acid and changes in light-scattering properties following fertilization of Urechis cuupo eggs. Dev. BioL 43, 299-312. PAUL, M. and GOULD-SOMERO, M. (1976). Evidence for a polyspermy block at the level of sperm-egg plasma membrane in Urechis caupo. J. Exp. Zoo1 196, 105-112. PRESLEY, R., and BAKER, P. F. (1970). Kinetics of fertilization in the sea urchin: A comparison of methods. J. Exp. BioL 52,455-468. RAVEN, C. P. (1966). “Morphogenesis: The Analysis of Molluscan Development.” Pergamon Press, Toronto.
of Mytilus galloprmincialis
33
REBHUN, L. I. (1962). Electron microscope studies on the vitelline membrane of the surf clam, Spisula solidissima. J. Ultrastruct. Res. 6,107-122. ROMANOFF, A. L. (1960). “The Avian Embryo.” Macmillan, New York. ROTHSCHILD, L. (1950). Counting spermatozoa. J. Exp. Biol. 26, 388-
395. ROTHSCHILD, L. (1956). “Fertilization.” Wiley, New York. ROTHSCHILD, L., and SWANN, M. M. (1952). The fertilization reaction in the sea urchin. The block to polyspermy. J. Exp. Biol. 29, 469483. VACQUIER, V. D., and PAYNE, J. E. (1973). Methods for quantitating sea urchin sperm-egg binding. Exp. Cell Res. 82,227-235. WOURMS, J. P. (1977). Reproduction and development in chondrychthyan fishes. Amer. Zoo1 17,379-410. ZIOMEK, C. A., and EPEL, D. (1975). Polyspermy block of Spisulo eggs is prevented by cytochalasin B. Science 189.139-141.
BIOLOGY
97,27-33
(1983)
Absence of a Complete Block to Polyspermy after Fertilization of /WyU/us galloprovincialis (Mollusca, Pelecypoda) Oocytes LOUISE Dummm-DuBk,**t “Station
FRANCOIS DuBb,**t PIERRE GUERRIER,*~’ AND PIERRE CouILLmDj-
Biologique, 29211 Roscoff, France; and TDkpartement des Sciences Biologiques, Universiti de Mont&al, C P. 6128, Mont&al, Q&bec, H3C 3J7 Canada Received July 1, 19X2; accepted in revised form December 1.3, 1982
Mytilus galloprovincialis oocytes undergo monospermic fertilizations (1 sperm nucleus/oocyte) over a wide range of sperm-oocyte ratios beyond which the number of penetrating sperm increases either linearly or exponentially over 10 min. Artificial activation of oocytes by KC1 or the ionophore A 23187, up to the polar body extrusion stage, allows successful fertilizations upon a subsequent insemination. No organized and complete detachment of supernumerary oocyte-bound sperm is detected after fertilization. Reducing the external Na+ concentration promotes a higher rate of fertilizations. These results suggest that no complete block to polyspermy is established in this species but that a partial block, Na+ dependent, might be sufficient to ensure monospermic fertilizations under natural conditions. INTRODUCTION
A successful fertilization results in the fusion of one male pronucleus to a female pronucleus and the further development of the zygote may then proceed normally. In species for which the penetration of more than one spermatozoon is the rule (physiological polyspermy), for example, pulmonate molluscs (Raven, 1966), urodeles (Fankhauser, 1948), elasmobranchs (Wourms, 1977), reptiles (Rothschild, 1956), and birds (Romanoff, 1960), monospermy is ensured at the level of the egg cytoplasm (for a review, see Austin, 1965). In most animal species, however, when more than one spermatozoon penetrate in the egg, the resulting polyspermy leads to the death of the embryo (Austin, 1965). To prevent the lethal effect of polyspermy, it is generally agreed that a specific defense mechanism, the so-called “block to polyspermy,” is set up by the egg to allow only one sperm to penetrate. In the well-studied sea urchin egg, the block to polyspermy appears to proceed in two distinct steps. First, a rapid partial block lowers the receptivity of the egg plasma membrane to further sperm fusions in less than 2-3 set after the first fertilization (Rothschild and Swann, 1952), this block being electrically mediated (Jaffe, 1976). One to two minutes after, this partial block is followed by a cortical granule exocytosis and the elevation of a fertilization coat around the egg which establishes a complete, but slow, block to polyspermy. However, the existence of a rapid partial block to polyspermy preceding the onset of the cortical reaction in sea urchin eggs has been and still is questioned by many 1 To whom reprint
requests should be addressed.
workers (Hagstriim, 1956; Byrd and Collins, 1975; De Felice and Dale, 1979;for a review, see Dale and Monroy, 1981). In the oocytes of the echiuran Urechis, a fast block to polyspermy begins to develop in less than 10 set after insemination (Paul, 1975a). Even though, in this species, surface coat reorganization occurs 4 min after fertilization, it has probably no role in polyspermy prevention (Paul and Gould-Somero, 1976). It has been later demonstrated that the fast partial block is directly related to a transient oocyte membrane depolarization, lasting around 10 min after fertilization (Gould-Somero et al., 1979). This is followed by a complete block of unknown nature at the level of sperm-egg plasma membrane fusion (Paul and Gould-Somero, 1976). In pelecypod mollusc oocytes, the cortical granules, though present, do not undergo exocytosis nor does any extracellular coat elevate upon fertilization (Pasteels and De Harven, 1962; Rebhun, 1962; Humphreys, 1967). Nevertheless, a complete block to polyspermy has been shown to take place in less than 15 set after fertilization (Ziomek and Epel, 1975) or after artificial activation (Longo, 1976) of Spisula oocytes. Our paper extends these earlier investigations to a second pelecypod mollusc, Mytilus galloprovincialis, and presents evidence that, contrary to Spisula, no complete block to polyspermy is established in the few minutes following fertilization or artificial activation of Mytilus oocytes. This result raises the question of the maintenance of monospermic fertilizations under natural conditions. A working hypothesis is presented to account for this original finding, an absence of complete block, and is also proposed as a suitable model to reconcile
27 0012-1606/83 $3.00 Copyright All rights
0 1983 by Academic Press, Inc. of reproduction in any form reserved.
28
DEVELOPMENTAL BIOLOGY
conflicting results on the fast block to polyspermy in sea urchin eggs. MATERIALS
AND
METHODS
Mytilus gallwpmvincialis were collected at Concarneau (Brittany) and kept in running seawater at the Station Biologique de Roscoff. Spawning was induced by placing the mussels in natural filtered seawater (FSW) at 30°C for 30 min, then in FSW at 4°C for 30 min. Spawning generally starts after returning the animals in FSW at room temperature. The sperm was collected “dry” and kept at 4°C until use. Working sperm suspensions were prepared by diluting the initial stock with FSW or artificial seawater (ASW) and sperm concentrations were determined turbidometrically at 680 nm, according to the equation: Y (sperm/ml) = 8.98 X OD X 10’. This equation was determined according to Rothschild (1950), by counting with a hemocytometer the number of sperm per milliliters in suspensions of various optical densities. The oocytes (naturally blocked at first metaphase of maturation) were washed several times with either FSW, ASW, or sodium-free seawater (NaFSW) acording to the experiments, and were kept at 15°C until use. Oocyte concentrations were determined by counting the number of oocytes in lo-p1 glass capillaries, taken from the working suspension. ASW was prepared according to the MBL formula (Cavanaugh, 19’75),with the addition of 2 mMTris (hydroxymethyl)-aminomethane. NaFSW had the same formula as ASW, except that NaCl was replaced by the molar equivalent of choline chloride. The pH was adjusted to 8.1-8.2 with NaOH for ASW and with KOH for NaFSW. Low-sodium seawater (LowNaSW) of variable final Na+ concentrations was prepared by mixing proper amounts of NaFSW and ASW. The incidence and severity of polyspermy were determined by counting the number of decondensed sperm nuclei in oocytes fixed 20 to 30 min after insemination in a 3:l mixture of ethanol:acetic acid for 1 hr, cleared in 55% acetic acid for 30 min, and kept in 70% ethanol (adapted from Byrd and Collins, 1975). Observations were done under phase-contrast microscopy, by counting 50-100 oocytes per sample. The time course of fertilizations was assayed by adding 0.001% to 0.01% sodium lauryl sulfate (SLS) to the gametes at various times after insemination (Hagstrom and Hagstriim, 1954). SLS inactivates all unbound sperm except those already fused with the oocyte plasma membrane (Byrd and Collins, 1975). In the case of Mytilus, this was tested by incubating sperm in different concentrations of SLS for 10 set, then adding these sperm to oocytes. No fertilization occurred above 0.0005% SLS;
VOLUME 97, 1983
to insure that at high-sperm concentrations, all unbound sperm were inactivated, higher concentrations were used, the eggs being washed 10 set after addition of the detergent. The time course of sperm-oocyte binding was determined by the subtraction method of Vacquier and Payne (1973), by inseminating a single batch of oocytes and by fixing aliquots at desired times in 1% glutaraldehyde in ASW. Artificial activation was achieved by adding either 10 PALM A 23187 (from a stock solution at 5 mM in 100% ethanol) or 20% v/v isotonic KC1 (0.52 M) to the oocyte suspensions for 1 hr. The percentage of activated oocytes was determined by counting the number of oocytes having extruded their polar bodies. The suspensions were then washed four times and inseminated. The percentage of fertilized oocytes was determined by counting the number of cleaved oocytes, 2 hr after insemination. All experiments were carried out at room temperature (18°C). Additional details are given in the text or figure legends. RESULTS
The relationship between the number of sperm penetrations and the sperm-oocyte ratio (Fig. 1) reveals that successful fertilizations (e.g., 1 sperm/oocyte) are obtained over a range of sperm-oocyte ratios between 10’ to 2 X 10’ (Table 1). Beyond these sperm-oocyte ratios, there is, at first, a monotonic increase in polyspermy (between 10zto lo3 sperm/oocyte) while over l5 X 103, the number of penetrating spermatozoa increases at an exponential rate. The sperm-oocyte ratios at which the change from monotonic to exponential increase occurs is very narrow (Fig. 5), and may vary, as mentioned above (l-5 X 103)from batch to batch. These observations are detailed on Table 1, where one can see that for different sperm densities, polyspermy increases in harmony with increasing sperm-oocyte ratios. Thus, monospermy is the rule only when the sperm-oocyte ratio is low, whatever the sperm density is. The number of penetrated spermatozoa for different sperm-oocyte interaction times, and ratios, was determined following the method of Byrd and Collins (1975). Examples of the three types of curve currently obtained are presented on Fig. 2. At low sperm-oocyte ratios, the first fertilization takes longer to be effected (it can be as long as 10 min) but the number of penetrating spermatozoa levels off at around 1 sperm/egg (Fig. 2A). When sperm-oocyte ratios are increased, the number of penetrating spermatozoa first tends to be in a linear relation with time (Fig. 2B) and, at higher ratios, tends to rise exponentially (Fig. 2C). These results demonstrate that over the time period tested (10 min), no complete block to polyspermy is established.
Fertilizatim
DUFRESNE-DUBS ET AL.
of Mgtilus
103
102
Sperm-oocyte
29
galloprovincialis
10
ratio
FE. 1. Induction of polyspermy by increasing sperm-oocyte ratios. Eighteen samples, 1 ml each, of ooeytes from one female, ranging from 4 X lo3 to 9 x 10” oocytes/ml, were inseminated with four different sperm densities, ranging from 1.86 X 10” to 9.38 X 10’ sperm/ml. Two minutes after mixing the gametes, the oocytes are washed and fixed 20 min after insemination. Sperm nuclei were counted in 50-100 randomly selected oocytes for each sample.
This is further suggested by the fact that oocytes artificially activated by KC1 or the calcium ionophore A 23187 and having extruded their polar bodies, are fertilizable upon subsequent insemination (Table 2). Observations of other KCl-treated oocytes revealed that TABLE 1 INDUCTION OF PQLYSPERMY BY INCREASING SPERM-O• CYTE RATIOS, FOR DIFFERENT SPERM DENSITIES Sperm Densities (sperm/ml) 1.87 x 106
1.87 X lo7
6.25 X lo7
9.38 x lo7
Sperm/oocyte
ratios
2.06 2.78 4.61 8.33 2.08
X X X x X
2.05 3.05 4.54 9.07 2.26
x lo*
1.03 1.37 2.06 4.12 1.03
10’ 10’ 10’ 10’ 102
x lo2 x 102
x 10’ X IO3
x lo3 x 103 X IO3 x lo3
x lo4
2.06 X lo3 2.68 X lo3
4.12 X lo3
Mean number of sperm/oocyte 1.05
1.08 1.34 1.43 1.53 2.06 2.31 3.67 4.62 19.46 3.55 4.62 6.98 13.06 50.10 3.67
13.98 21.10
more than 80% of oocytes contain at least one sperm pronucleus after insemination. This observation provides evidence that cleavages reported in Table 2 involved sperm penetrations and were not mere “parthenogenetic activation” without sperm fusion and incorporation. The extrusion of polar bodies normally takes place 20-25 min after fertilization at 20°C. Sperm-oocyte binding assays (Fig. 3) reveal that sperm bind mostly during the first 15 see after insemination, but that no full detachment will occur over the following 10 min. The effect of lowering the external Na+ concentration for different sperm-oocyte ratios (Fig. 4) shows that, for low ratios, a 20 mM external Na+ concentration, or below, induces a small increase of polyspermy (mean number of sperm/oocyte). However, higher sperm-oocyte ratios, only slightly polyspermic at normal Na+ concentrations (425 mM), induce a four- to fivefold increase in the number of penetrated sperm when the external Nat concentration is below 300 mM. These results suggest the possibility of a cumulative negative effect on the oocyte defense mechanism when high sperm-oocyte ratios and low-sodium seawater are used at the same time. DISCUSSION
Even though polyspermic fertilization of M&ilus oocytes leads to abnormal cleavages and eventually to the death of the embryo, our results demonstrate that no
30
DEVELOPMENTAL BIOLOGY
VOLUME 97, 1983 TABLE 2 FERTILIZATION OF ARTIFICIALLY ACTIVATED Mytilus
OOCYTES
12-
Treatment
Percentage of oocytes having extruded first polar body after 90 min”
Percentage cleavages of treated oocytes 120 min after inseminatior?
A23187 (10 @f) KC1 (20%, v/v)
96.3 95.5
90.2 98.8
11 -
lo-
0
I
Note. Mean results of two experiments with two batches of oocytes. a None of the unstimulated control oocytes or oocytes submitted to 0.2% ethanol extruded polar bodies. * None of the KC1 or A23187-activated oocytes cleaved without insemination. Additional experimental procedures are given under Materials and Methods.
2-
I
Time
t
1
I
I
I
I
I
n
Spi.sula oocytes are at the germinal vesicle stage (prophase I). This difference in the physiological conditions of Spisula and Mytilus oocytes has been related to the fact that Spi.sula oocytes release acid upon fertilization (Guerrier et al., 1981) while Mytilws oocytes do not (Paul, 19’7513; Dub6 and Guerrier, 1982). The present paper adds new results to the growing list of physiological differences between Spisula and Mytilus oocytes. How can an animal species resist the lethal effect of polyspermy without an absolutely efficient defense mechanism? In an attempt to answer this question and according to our results, a schematic working hypothesis is presented (Fig. 5). Under reasonable sperm-oo-
5 6 7 8 9 10 of transfer in SLS (min.)
FIG. 2. Severity of polyspermy for different sperm-oocyte interaction times. A single batch of oocytes is inseminated at 0 min. At the indicated times, oocytes are transferred into 0.901% SLS (A) or 0.01% SLS (B and C) for 3 min, washed, and fixed 20 min after insemination. Final sperm-oocyte ratios: (A) 1.2 X 10’; (B) 3.0 X 103; (C) 4.4 x los.
specific defense mechanism completely protects the oocyte from this “deleterious” possibility. This result is consistent with the fact that, upon fertilization of i@tilus oocytes, no extracellular coat elevates which, in turn, would form an absolute barrier to further sperm penetrations as is the case for sea urchin eggs. However, our results contrast those obtained for Spi&a oocytes which establish a complete block to polyspermy in less than 15 set after the first fertilization (Ziomek and Epel, 1975). Moreover it has also been shown that Spisula oocytes artificially activated by a treatment with hypertonic seawater (Longo, 1975) cannot be subsequently fertilized. This differs from our results on KC1 and A23187 activations (Table 2). It must be kept in mind that, at the time of fertilization, M@ilus oocytes are arrested at the metaphase I of meiotic maturation while
i 800-
o
1
2 Time
3 4 5 of transfer
6 7 8 9 IO in glutaraldehyde(min)
FIG. 3. Time course of sperm-oocyte binding. A single batch of oocytes is inseminated with the same volume of a sperm suspension. At the indicated times, 2 ml of gametes is transferred into 2 ml of 2% glutaraldehyde in seawater. The optical density of the supernatant is then measured. The number of bound spermatozoa is obtained by calculating the difference between the total number of spermatozoa available and the number of unbound sperm. It is then divided by the number of oocytes per milliliter. Final sperm-oocyte ratios: (0) 3.97 x 10s; (0) 4.95 x lo?
Fertilization of
DUFRESNE-DUBS ET AL.
mc z
. O
n
n 100
’
’ 200
.
1 300
Na+Concentration
. 400
(mM)
FIG. 4. Induction of polyspermy at different centrations. Oocytes were incubated in 10 ml of NaSW for 15 min, and inseminated. Interaction for 20 min, and then the oocytes were fixed. Final (A) 1.69 X 10’; (B) 9.89 X lo*; (C) 1.77 X 103.
external sodium conASW, NaFSW, or low of gametes occurred sperm-oocyte ratios:
cyte ratios, monospermy is the rule while excessive sperm-oocyte ratios result in “explosive” polyspermy. This could imply that a partial block to polyspermy ensures monospermic fertilizations under sperm-oocyte
OQCYTE DEFENCE AGAINST POLYSPERMY
Mytilus
galloprcn+xialis
ratios likely to reflect the situation prevailing under natural conditions. Under experimentally imposed conditions (excessive sperm-oocyte ratios), the efficiency of this partial block to polyspermy rapidly decreases down to a level at which it becomes completely overwhelmed. A decrease in egg receptivity to spermatozoa as soon as the first fertilization is effected would lower the probability of a successful1 second fertilization. This hypothesis was first proposed by Rothschild and Swann (1952) for the sea urchin egg. If one assumes that this first partial block is related to a transient membrane depolarization (Jaffe, 1976) it appears likely that the efficiency of this partial block would fluctuate with time since such an initial membrane depolarization is followed up by a gradual hyperpolarization in all the marine invertebrate eggs so far studied (Hagiwara and Jaffe, 1979). The relative efficiency decrease of the oocyte defense mechanism may also be related to the cumulative effect of extruded sperm lysins digesting the oocyte extracellular coats which appear likely to play a determinant role in the protection against polyspermy (Hagstrom, 1956; Dale and Monroy, 1981). On the other hand, an increase of the sperm concentration is also likely to result in a higher “collision rate” between sperm and oocytes.
FERTILIZING ACTIVITY
SPERM-OOCYTERATIO
J2 $8 "6
a
? 1
ATTAIN A PLATEAU AT 1 SPERM PER OOCYTE
LINEAR
(NATURAL CONDITIONS ?) FIG. 5. Schematic
working
hypothesis
31
EXPONENTIAL
(LABORATORY IMPOSED CONDITIONS ?) on the block to polyspermy
in Mytilus
oocytes.
32
DEVELOPMENTAL BIOLOGY
Such an antagonistic effect of raising sperm-oocyte ratios on the oocyte defense mechanism and on the sperm fertilizing activity (Fig. 5) would explain the rapid increase of fertilization rates depicted in Figs. 1 and 2. In ascidian eggs which also lack a cortical reaction, Lambert and Lambert (1981) have shown, by the method of a first monospermic insemination, followed at various time intervals by a second polyspermic insemination, that these eggs could be incapable of refertilization by a second spermatozoon about 21 see after the first fertilization. However, Lambert and Lambert (1981) further mentioned that the half-conduction time for the establishment of this block to polyspermy could require more than 5 min when higher sperm concentrations were used. With Myths oocytes, we have also obtained, by the same method, highly variable estimations of the conduction time of the establishment of the block to polyspermy, ranging from 2 min to the complete absence of any block to polyspermy by 10 min, depending on the sperm-oocyte ratios used (data not shown). Lambert and Lambert (1981) claimed that the timing for the establishment of the block to polyspermy in ascidian eggs is dependent upon the sperm concentration. This seems to us the indication of an incomplete partial block fitting well with our proposed model (Fig. 5). The involvement of extracellular Naf in the maintenance of a lower fertilization rate (Fig. 4) supports the hypothesis that Na+-dependent membrane depolarization is related to a partial block of Mytilus oocytes as is the case in Ascidians (Lambert and Lambert, 1981) and in Urechis for which it has been demonstrated that low-sodium seawater reduced the fertilization potential level, thus allowing supernumerary spermatozoa to penetrate (Gould-Somero et al., 1979). Whatever the exact nature of this block is, we suspect a possible similarity with the presumed fast block to polyspermy of sea urchin eggs. Measurements of fertilization rates in sea urchin eggs, determined by counting sperm nuclei, reveal either a rate of second sperm penetration lower than the rate of first sperm penetration (Presley and Baker, 1970) or a linear rate of sperm penetrations strictly dependent upon the sperm-egg ratio (Byrd and Collins, 1975). A careful examination of their data may lead one to interpret the rate of sperm penetrations, at the highest sperm-egg ratio tested, as being exponential rather than linear (Fig. 3A of Byrd and Collins (1975)). These three types of observations roughly correspond to our results (Fig. 2) which stress the fact that fast partial blocks to polyspermy might not be processes resulting in a fixed decrease of the egg membrane receptivity to spermatozoa. Therefore, previous determinations of sea urchin egg fertilization rates might not be contradictory as would seem at first sight, but
VOLUME 97, 1983
would reflect a real, though complex, situation. The main problem in studying the fast partial block to polyspermy in sea urchin eggs is that it is experimentally difficult to separate its possible role from the obvious one of the cortical reaction which may begin as soon as 20 set after the first fertilization. To understand better the nature of these partial blocks to polyspermy, Mytilus oocytes might therefore provide a new, promising material for investigation. Also promising would be investigations on the precise nature of the complete blocks to polyspermy in Urechis (Paul and Gould-Somero, 1976) and Spisula oocytes (Ziomek and Epel, 1975). Mrs. Claude Guerrier is acknowledged for drawing the figures and Mrs. Nicole Guyard for typing the manuscript. We also deeply thank Drs. Michael J. Whitaker and Brian Dale for valuable comments on the first draft of this paper. Thanks are due to an anonymous reviewer for helpful suggestions and requests. This work was supported by FCAC (Quebec) Ph.D. scholarships to L.D.D. and F.D., by a DGRST (France) grant (ACC 79.70777) to P.G. and a NSERC (Canada) grant to P.C. REFERENCES Prentice-Hall. Englewood Cliffs, AUSTIN, C. R. (1965). “Fertilization.” New Jersey. BYRD, E. W., and COLLINS,F. D. (1975). Absence of fast block to polyspermy in eggs of sea urchin Strungylocentrotus purpuratus. Nature (London) 257, 675-677. CAVANAUGH,G. M. ed. (1975). “Formulae and Methods VI of the Marine Biological Laboratory.” Woods Hole, Mass. DALE, B., and MONROY,A. (1981). How is polyspermy prevented? Gamete Res. 4, 151-169. DE FELICE, L. J., and DALE, B. (1979). Voltage response to fertilization and polyspermy in sea urchin eggs and oocytes. LIev. Biol. 72,327341. DUB& F., and GUERRIER, P. (1982). Acid release during activation of Barnes can&i& (Mollusca, Pelecypoda) oocytes. Deu. Growth &fferen. 24.163-171. FANKHALJSER, G. (1948). The organization of the amphibian egg during fertilization and cleavage. Ann N. Y. Awd Sci. 82.684-708. GUERRIER, P., DUB& F., and MOREAU, M. (1981). External calcium requirements for oocyte maturation in the surf clam Spisula solidissimu Biol Bull. 161, 335-336. GOULD-SOMERO, M., JAFFE, L. A., and HOLLAND, L. (1979). Electrically mediated fast block in eggs of the marine worm Urechis ~aupu..J.
Cell BioL 82.426-440. HAGIWARA, S., and JAFFE, L. A. (1979). Electrical properties of egg cell membranes. Annu. Rev. Biophys. Bioeng. 8,385-416. HAGSTR~M,B. E. (1956). Studies on polyspermy in sea urchins. Arkiv. ZOOL 10,307-315. HAGSTR(JM, B., and HAGSTROM,B. (1954). The fertilization rate in sea urchins. Exp. Cell Res. 6,479-484. HUMPHREYS,W. J. (1967). The fine structure of cortical granules in eggs and gastrulae of Mytilus edulis J. Ultra&-u&. Res. 17, 314326. JAFFE, L. A. (1976). Fast block to polyspermy in sea urchin eggs is electrically mediated. Nature (London) 261, 68-71. LAMBERT,C. C., and LAMBERT, G. (1981). Formation of the block to polyspermy in ascidian eggs: Time course, ion requirements, and role of the accessory cells. J. Exp. Zool. 217, 291-295. LONGO, F. J. (1976). Ultrastructural aspects of fertilization in spiralian eggs. Amer. Zoo1 16,275-394.
DUFRESNE-DUBI? ET AL.
Fertilization
PASTEELS, J. J., and DE HARVEN, E. (1962). Etude au microscope electronique du cortex de l’oeuf de Barnea candidu (Mollusque bivalve), et son evolution au moment de la fecondation, de la maturation, et de la segmentation. Arch Biol. (Liege) 73,465-490. PAUL, M. (1975a). The polyspermy block in eggs of Urechis caupo. Evidence for a “rapid” block. Exp. Cell Res. 90.137-142. PAUL, M. (1975b). Release of acid and changes in light-scattering properties following fertilization of Urechis cuupo eggs. Dev. BioL 43, 299-312. PAUL, M. and GOULD-SOMERO, M. (1976). Evidence for a polyspermy block at the level of sperm-egg plasma membrane in Urechis caupo. J. Exp. Zoo1 196, 105-112. PRESLEY, R., and BAKER, P. F. (1970). Kinetics of fertilization in the sea urchin: A comparison of methods. J. Exp. BioL 52,455-468. RAVEN, C. P. (1966). “Morphogenesis: The Analysis of Molluscan Development.” Pergamon Press, Toronto.
of Mytilus galloprmincialis
33
REBHUN, L. I. (1962). Electron microscope studies on the vitelline membrane of the surf clam, Spisula solidissima. J. Ultrastruct. Res. 6,107-122. ROMANOFF, A. L. (1960). “The Avian Embryo.” Macmillan, New York. ROTHSCHILD, L. (1950). Counting spermatozoa. J. Exp. Biol. 26, 388-
395. ROTHSCHILD, L. (1956). “Fertilization.” Wiley, New York. ROTHSCHILD, L., and SWANN, M. M. (1952). The fertilization reaction in the sea urchin. The block to polyspermy. J. Exp. Biol. 29, 469483. VACQUIER, V. D., and PAYNE, J. E. (1973). Methods for quantitating sea urchin sperm-egg binding. Exp. Cell Res. 82,227-235. WOURMS, J. P. (1977). Reproduction and development in chondrychthyan fishes. Amer. Zoo1 17,379-410. ZIOMEK, C. A., and EPEL, D. (1975). Polyspermy block of Spisulo eggs is prevented by cytochalasin B. Science 189.139-141.