J. Exp. Biol. (1970), 53, 455-4681 plate and 8 text-figures

inted in Great Britain

KINETICS OF FERTILIZATION IN THE SEA URCHIN:A COMPARISON OF METHODS

BY R. PRESLEY

Department of Anatomy, University of Cambridge*

AND P. F. BAKER

Department of Physiology, University of Cambridge

(Received 7 November 1969)

INTRODUCTION

In order to study the rate of fertilization it is necessary to employ an agent whichwill both achieve a rapid functional separation of sperm from eggs and also allow theuse of some method of assessing the numbers of unfertilized, fertilized and poly-spermic eggs in a sample. Since present methods do not allow of a distinction at the timeof fertilization between fertilizing sperm and others at the egg surface, all spermicidesused must permit the eggs to remain viable long enough for the successful sperm toundergo some recognizable developmental change. In previous work the extent offertilization has been assessed at first cleavage, and the only spermicides which havebeen used extensively are dilute sea water (Rothschild & Swann, 1952) and sea watercontaining traces of the detergent lauryl sulphate (Hagstrom & Hagstrom, 1954;Allen & Griffin, 1958). These two methods do not seem to have been compared on thesame batch of eggs, which is surprising in view of reported differences both in thedetailed shape of the fertilization rate curves and in their interpretation (Runnstrom,1961). In this paper we report a comparison of these and other spermicides using amethod of assessing fertilization based on counting male pronuclei. This method wasmentioned in a preliminary report (Baker & Presley, 1966) and has been found toprovide a more accurate measure of the degree of polyspermy than does the cleavagemethod.

MATERIALS AND METHODS

Preparation of eggs and sperm

Mature specimens of Echinus esculentus were obtained at Plymouth in late Marchand early April and kept in sea-water tanks for a few days until use. Mature specimensof Psamtnechinus miliaris were obtained from the Wash in late July and early August,transported to Cambridge and kept until use in an indoor sea-water aquarium withcontinuous circulation and aeration. Before use, specimens were rinsed for 30 sec.with 30% artificial sea water (A.S.W.) to destroy any free spermatozoa and thenwashed with normal sea water. Release of eggs and sperm was induced by hemisectingthe specimens and standing the gonad-containing half-shell over a collecting vessel.Eggs were shed into filtered natural sea water (E. esculentus) or artificial sea water

• Present address: Department of Anatomy, University College, Cathays Park, Cardiff CFi 3NR.

456 R. PRESLEY AND P. F. BAKER

(P. miliaris), and kept at the desired experimental temperature before use. Sperm waa|shed into a beaker surrounded by ice, containing a small quantity of a solution which'was half molar with respect to both dextrose and glycine. Stock sperm so obtained waskept at o° C.

After collection eggs and sperm were tested for fertilization, and batches giving lessthan 95 % fertilization or containing more than 5 % eggs in the germinal vesicle stagewere rejected. Before each experiment eggs were washed in A.s.w., and allowed tosettle before re-washing; all eggs were washed twice, and at the second washing weredivided among test tubes so that each sample used throughout the experiment wasrepresentative of the whole population of eggs. Where pre-treatment with any agentwas employed, the supernatant was removed and the treatment fluid was added foran appropriate period prior to the experiment. All solutions to which the eggs wereexposed were kept with the eggs in a water bath at the experimental temperature,usually 160 C.

The density of the stock sperm solution was determined with a haemocytometerslide, after dilution with distilled water. Immediately before using sperm for a seriesof fertilizations, the stock suspension was agitated thoroughly and then diluted withA.s.w. to give the required sperm density. This diluted sperm was then kept at theexperimental temperature.

Solutions

The composition of A.S.W. was NaCl, 460 mM; KC1, 10 mM; MgClj,, 55 mM;CaCl2, 11 mM; NaHC03, 2-5 mM; pH 8-o.

The spermicidal fluids generally used (and the abbreviations used throughout thispaper) were:

30-A.s.w.: 30% artificial sea water as above, pH 8-o.Lauryl sulphate: 0-002% in A.S.W. pH 8-o.KC1: A.s.w. in which the NaCl was replaced isosmotically with KC1, pH 8-o.EDTA: 100 mM-Na-EDTA; 300 mM-NaCl; 10 mM-KCl adjusted to pH 8-o with

NaHC03.UN03: A.s.w. containing 0-3 mM uranyl nitrate (UOa(NO3)2, 6H2O), pH 6-3.

This solution was prepared from a stock solution of 10 mM uranyl nitrate in distilledwater by adding this to an appropriate quantity of A.s.w. Such a solution did not pre-cipitate, though at higher molarities and at more alkaline pH a precipitate formed.

Nicotine (British Drug Houses) was freshly prepared before each experiment asstock solution of 5 drops (200 nig.) per 100 ml. A.S.W., pH 8-o (approximately12-5 mM), and appropriately diluted before use.

Choice and efficacy of spermicides

In preliminary experiments spermicidal agents were tested for their effect on spermmotility, and subsequently for their ability to allow further development of fertilizedeggs. None of the agents abolished sperm motility completely, less than 1 % of spermremaining feebly motile for several minutes. The experimental procedure was there-fore designed to effect a 1/100 dilution of sperm at the time of inactivation withspermicide. Uranyl nitrate was included because of Okazaki's (1956) report thaturanyl ions reversibly block fertilization without causing any immediate reduction in

Kinetics of fertilization in the sea urchin 457

iperm motility. Preliminary results confirmed this report and uranyl nitrate wastherefore compared with the other agents.

Each rate-determination experiment included a sample of eggs placed unfertilizedin 15 ml. of spermicide, followed after 15 sec. by the addition of a quantity of spermtwice as great as that present in samples from the corresponding rate experiment.Such a sample was taken as a 'zero' time point for each experiment, the procedurestemming from the practical difficulty of mixing eggs, sperm and spermicide thoroughlyand simultaneously. 30-A.s.w. was the only spermicide which repeatedly failed toblock fertilization in 100% of the eggs. With sperm densities of io7 and 10s sperm/ml.,the percentage of eggs fertilized in the presence of 30-A.s.w. averaged 22 ± 8 and40 ± 6 % respectively.

A further measure of the effect of the spermicides was obtained by measuring the oxy-gen consumption of sperm in the presence of the agents. The oxygen consumption wasreduced to less than 10 % of its control value by those agents which immobilized sperm.

Fertilization

1-5 ml. of eggs was placed in a 5 ml. beaker in a water bath at the desired experi-mental temperature, and agitated by a small glass-covered stirrer for about 3 sec.before and after addition of sperm.

At the desired time intervals after addition of sperm, samples of the sperm-egg mix-ture were decanted into 30 ml. of spermicide, pre-cooled to the temperature of furtherdevelopment, which had been kept gently agitated in a 250 ml. beaker. In all experi-ments zero time was taken as the time of adding sperm to the eggs, and duration offertilization as the time up to decanting into spermicide. After addition of the sampleto the spermicide the mixture was rapidly transferred to a boiling tube, except in thecase of 30-A.s.w. where the sea water was first rendered isosmotic after 30 sec. by theaddition of 10 ml. of hypertonic (3 x isotonic) sea water. The tubes were placed in awater bath for 10 min. at 150 C. (E. esculentus), or for 10 min. at io° C. (P. miliaris),and the eggs were left to develop. It should be noted that with most of the agents somesperm recover on return to normal sea water, and to avoid errors it is necessary forfurther development to take place in the inactivating fluid.

Scoring of fertilization

At the end of the development period a sample of eggs was withdrawn from thebottom of the boiling tube with a pipette of mouth bore 1 mm. diameter and placedon a 3 in. x 1 in. microscope slide. A f in. square microscope slide, supported at eachcorner by a globule of Cow Gum (P. B. Cow (Li-Lo), Slough, Bucks, England), waslowered over the sample and pressed down until the eggs were lightly gripped. Theslide was then placed in 25 % acetic acid, 75 % absolute alcohol for 24 hr. To avoidfixation artifacts it was necessary to use fresh fixative for each experiment.

After fixation the slides were placed in 0-5 % orcein in 45 % aqueous acetic acidfor 2-3 hr., and then the staining solution was replaced with 10% aqueous acetic acidand the edges of the coverslip were sealed with molten paraffin wax. Preparationscould be kept for several weeks without deterioration in a sealed chamber over 10%acetic acid.

3O E X B 5 2

458 R. PRESLEY AND P. F. BAKER

Plate i shows eggs stained in this manner. Pronuclei were counted with a xobjective using light-ground phase-contrast optics which enabled visible expansion othe pronucleus, penetration below the surface of the egg, and the presence of an associ-ated sperm aster to be used as distinguishing criteria. Routinely, 25 eggs in a samplewere counted sequentially on the slide, starting at a randomly selected point; in someexperiments 100 eggs from each sample were counted.

In preliminary experiments the cleavage method of assessing fertilization was com-pared with counts of male pronuclei. After withdrawing a sample of eggs for micro-scopy the spermicide was decanted off the remainder, which was placed in a beakerin an excess of filtered natural sea water in which further development took place at17° C. An assessment was made of fertilization by the morphology of the first cleavage

1001- (A)

• a50

100 1- (8)

0 50 100% fertilized (sperm count)

0 50 100% polyspermic (sperm count)

Text-fig. 1. Relationship between cleavage and pronuclear counts. (E. esculentus). Each pointis the percentage of normal eggs fertilized (A), or polyspermic (B), as judged by the cleavagemorphology of a sample of 200 eggs examined at 50 x under a binocular dissecting microscope(ordinate); or as found by pronuclear count on 25 eggs drawn from the same experimentalgroup (abscissa).

division. Text-fig. 1 shows the results of a comparison between cleavage counts andpronuclear counts for total fertilization and polyspermic fertilization. A linearcorrelation is apparent for total fertilization, though the scatter is wider than ideallyexpected. Where more than 20% of the eggs are polyspermic, pronuclear countsconsistently give a higher score than does cleavage. With nicotine pre-treatment a highdegree of polyspermy can be attained in all eggs (Clark, 1936), but at the time whencontrol eggs are undergoing first cleavage, such polyspermic eggs are indistinguish-able from unfertilized eggs except for the presence of a fertilization membrane (PI. 1D, E). Subsequently these eggs become irregular and divide into multiple cytoplasmicfragments when control eggs are at the 8 to 16-cell stage. A few of these eggs developinto swimming gastrulae of abnormal appearance. It was found to be impossible toscore the degree of polyspermy in such eggs with accuracy by the cleavage methodand the impression was gained that this difficulty was present in any egg with morethan four male pronuclei.

Kinetics of fertilization in the sea urchin 459

Analysis of sperm distribution

In samples where 100 eggs were scored the distribution of sperm among the eggswas compared with theoretical distributions calculated from the following probabilityconsiderations. Each sample represents a number of eggs exposed to a large excessof sperm for the same time. The number of sperm entering the eggs is small by com-parison with the total number of sperm present. If sperm entry is at the same rateregardless of whether or not the egg has already been fertilized or re-fertilized, theresulting distribution of male pronuclei should follow a Poisson distribution:

Px = (i*»/*l) e~m,

where Px is the probability of occurrence of a given degree of polyspermy, x; m isdenned as

total number of male pronuclei in a sample of eggstotal number of eggs in the sample

In a sample of iV eggs, the number of eggs with x male pronuclei is given by

The value of m may be calculated directly from the sample or from the numbers ofeggs remaining in the initial condition, n^.

«o = Ne-m,

from which m = In (N/n^).

If a large deviation from a Poisson distribution is expected, the latter method is prob-ably a better way of estimating m from the sample.

Distribution functions may be calculated either taking the unfertilized eggs as theinitial condition, in which case a deviation from the expected Poisson distribution willrepresent a departure from the initial fertilization rate for re-fertilization; or theunfertilized eggs in the sample may be discounted, and the monospermic eggs takenas the initial condition for the calculation of a distribution, deviation from which mayrepresent a change in the rate of re-fertilization with successively more sperm enteringthe egg. It should be noted that in this latter case the theoretical distribution will onlybe approximate, since in practice the monospermic eggs to be re-fertilized do not appearsimultaneously, and hence not all eggs have been susceptible to polyspermy for thesame length of time.

RESULTS

Comparison of fertilization rates obtained with different agents

The degree of fertilization can be expressed either as the percentage of eggs fertilizedor as the mean number of male pronuclei (sperm) per egg, for any sample. The formergives a measure of the rate of fertilization, while the latter provides extra informationabout the degree of polyspermy. Results obtained with different spermicides on thesame batch of eggs of E. esculentus are shown in Text-fig. 2 A, B.It can be seen that thecurves fall into three types: (1) 30-A.S.W. which gives a high initial rate of fertilizationwith a large incidence of polyspermy in the period from 15 to 40 sec, (2) uranylnitrate which shows no apparent fertilization until after 15 sec, but at long timeintervals shows the same degree of fertilization as the other agents; (3) KC1, EDTA,

30-i

460 R. PRESLEY AND P. F. BAKER

and lauryl sulphate, all of which are essentially similar in giving an apparent increasdin rate during the first 15 sec., with the rate subsequently declining. Essentiallysimilar results were obtained with P. mtliaris, a marked lag in fertilization being

100r

Mean sperm/egg

20

10

10 20 30Time (sec.)

40 20 40 60Time (sec)

Text-fig. 2. Comparison of inactivating agents {E. esculentus). (A) Fertilization rate curvesfor 30-A.8.W. (•); EDTA ( x ); UNO, (O); each point the mean ± s.E. of mean of countson 25 eggs from batches taken from four separate experiments. Curves for KC1 (A); laurylsulphate (•) ; from single experiments, each point from a count of 100 eggs per sample.(B) Mean number of male pronuclei (sperm) per egg plotted against time for 30-A.s.w. (•);EDTA ( x ) ; KC1 ( A); UNO, (O). Single experiment, each point from count of 25 eggs.Conditions for both A and B: sperm density at fertilization 5x10' sperm/mL Temperature15° C. Sperm added at zero time.

Mean sperm/egg

<A\ 60

1 0 0 r

20 -40 60 80Time (sec.)

180 10 20 30Time (sec)

Text-fig. 3. (A). Comparison of inactivating agents (P. mtliaris). Fertilization rate curves for30-A.s.w. (•) ; KC1 (A); UNO, (O). Single experiment, each point from counts of 25 eggs.1*4 x io8 sperm/ml. Temperature io° C. Sperm added at zero time. (B) Comparison of inacti-vating agents on nicotine-treated eggs (E. esculentus). Eggs stood for 10 min. in A.s.w. containingia-5 mM nicotine (pH 8-o), and were transferred in that solution to the fertilization chamber.Single experiment, each point from a count of 25 eggs. Curves for 30-A.s.w. (•) ; KC1 (A);UNO, (O). Temperature 15° C. Sperm density 10' sperm/mL Sperm added at zero time.

Kinetics of fertilization in the sea urchin 461

apparent with UNO3, although in this species the curve obtained with 30-A.s.w. moreclosely approached those of the other spermicides in the KC1 group (Text-fig. 3).

lnactivation with 30-A.s.w.

It was evident from the preliminary experiments on efficacy of kill (see Methodssection) that at high sperm densities 30-A.s.w. was not able to produce completeinactivation of sperm at the instant of application. It may therefore be suspected thatthis is one factor contributing to the apparently higher rate of fertilization at shorttime intervals shown by 30-A.s.w. in comparison with the other agents. A second effect of

Table 1. Degree of polyspermy in comparable samples of 100 eggs withdifferent spermicides

S sec. 15 sec

Agent

30-A.s.w.Lauryl sulphateKC1

Unfertil-ized

368988

Mono-spermic

Siir12

Poly-spermic

1300

Unfertil-ized

27S°5°

Mono-spermic

4242

44

Poly-spermic

3186

Samples from same experiment. Sperm density io7 sperm/ml. Temperature i6° C.

70

Sg 60o

I" 50

£ 40rt

Z 30

20

10

0 10 20

Time (sec.)

30 40

Text-fig. 4. Promotion of polyspermy by 30-A.s.w. on nicotine-treated eggs at high spermdensities (E. esculentus). Eggs stood for 10 min. in A.8.W. containing 12-5 mM nicotine (pH 8-o),and were transferred in that solution to the fertilization chamber. Sperm densities at fertiliza-tion indicated on curves. (•). Control eggs (open circles) fertilized at sperm density io*sperm/mL Inactivation with 30-A.s.w. Single experiment, counts of 25 eggs. Temperature170 C. Sperm added at zero time.

30-A.s.w. is the promotion of polyspermy. This phenomenon is evident in Text-fig. 2B.With E. esculentus comparison of the sperm distribution in egg samples fertilized for thesame time intervals at the same sperm density showed that with 30-A.s.w., especiallyat short time intervals, there was always present a greater number of polyspermic eggs

462 R. PRESLEY AND P. F. BAKER

than with the other agents (Table 1). At high sperm densities it was often found thatsamples where the sperm were inactivated in the time intervals from 5-40 sec. showeda greater degree of polyspermy than did samples from the same experiment but atlater times. With eggs pre-treated with nicotine, which promotes a high degree ofpolyspermy (Hertwig & Hertwig, 1887; Clark, 1936; Rothschild & Swann, 1950;Baker & Presley, 1966), this polyspermy-promoting effect of 30-A.s.w. at high spermdensity is very marked (Text-fig. 4).

40 50 60

Time (sec)

Text-fig. 5. Effect of temperature on fertilization rates in KC1 and UNO3 (P. miliaris).Fertilization rates as determined with KC1 (•); UNO, (O); both at pH 6-3. Upper curvesio° C, lower curves 20 ° C. Single experiment, counts on 25 eggs. Sperm density 17X 10'.Sperm added at zero time. Eggs cooled or wanned to experimental temperature from 160 C.progressively over 15 min.

Such an effect was not found where the other agents were employed to inactivatethe sperm. In the case of P. miliaris, the polyspermy-promoting action of 30-A.s.w.was much less marked, a significant excess of polyspermy at short time intervals overlong time intervals never being obtained, although the initial rate of fertilization asdetermined with 30-A.s.w. was always apparently higher than with the other agents.

Inactivation with uranyl nitrate

In both P. miliaris and E. esculentus inactivation of sperm with uranyl nitrate-containing sea water produces rate curves which differ from those obtained with theother agents in that there is a marked lag before fertilization becomes apparent.A similar difference between uranyl nitrate and the other spermicides is seen in thecase of eggs treated with nicotine prior to fertilization (Fig. 3 E). After nicotine treat-ment a marked degree of polyspermy is attained with all agents at time intervalsgreater than 40 sec, but at intervals up to 15 sec. UNO3-inactivation gives unfertilizedeggs, while the other agents yield considerable polyspermy.

The nature of the difference between the UNO3 rate-curves and the others was

Kinetics of fertilization in the sea urchin 463

^investigated in more detail in a comparison between KCl and UNO3 on normal eggs.The separation between the curves is temperature-sensitive (Text-fig. 5), temperaturehaving a somewhat greater effect on the UNO3 curve (£?io-2o = 5"2) t^1 1 o n t n e KClcurve (P10-20 = 3' 6)- The KCl in routine use differs from UNO3 in pH (KCl,pH = 8-o; UNO3, pH = 6-3) as well as in ionic composition. When K-rich A.S.WpH 6-3, was compared as an inactivating agent with the same solution containingUNO3, the separation between the rate curves was still present, and the KCl curvewas very similar to that obtained with the K-rich A.s.w. in routine use. The differencebetween the curves was abolished when uranyl nitrate A.s.w. to which had been added5 mM EDTA without change of pH was employed as the inactivating agent. Thus itmay be concluded that the uranyl ions are responsible for the differences between thecurves. Eggs which have been fertilized for 15 sec. and then inactivated with K-richA.s.w. containing uranyl nitrate and allowed to develop in that agent show no evidenceof fertilization; however, if after short periods of inactivation in that agent the eggsare transferred to uranyl-free K-rich A.s.w., a number of eggs develop male pronuclei(Baker & Presley, 1969). This suggests that K-rich A.s.w. alone is unable to maintainthe block on pronuclear development produced by UNO8 and indicates that the twoagents act at different stages in the process, uranyl ions acting later than potassium.

Inactivation with other agents

With KCl, EDTA, and lauryl sulphate as the inactivating agents a rate curve ofsigmoid shape is obtained, indicating a lag in fertilization rate in the initial 5 sec. Thislag is followed by a rapid increase in fertilization rate and then a progressive declineat time intervals when most of the eggs are fertilized. In the case of E. esculentus, thecurve as determined with EDTA often showed a slightly lower degree of fertilizationthan did KCl and lauryl sulphate at the same time intervals; this was not the case withP. miliaris, and the difference may not be significant as the microscopic appearance ofthe E. esculentus eggs showed surface damage with EDTA, and errors of scoring maytherefore be present. It was concluded that KCl and lauryl sulphate were the mostsatisfactory agents for determinations of fertilization rate, as they acted rapidly butdid not produce a spurious polyspermy at short time intervals. However, the sigmoidshape of the curves makes impossible accurate calculation of the initial rate of fertiliza-tion for comparison with re-fertilization, after the manner of Rothschild & Swann

Effect of the jelly coat

One possible explanation of the initial lag retardation of the sperm penetration by thejelly coat. The jelly coat of samples of eggs was removed by gentle shaking in A.s.w.,pH 5-3 (Hagstrom, 1956), until the eggs were free of jelly as judged by microscopicexamination of the packing of a sample in a watch-glass and the absence of trappingof sperm in the jelly coat of the eggs in this sample on test fertilization. A comparisonof fertilization rates of such jelly-free eggs with those of normal eggs from the samefemale, using KCl and lauryl sulphate as inactivating agents, showed that in theabsence of jelly fertilization was more rapid but the sigmoid nature of the curvewas still evident (Text-fig. 6 A). Similar results were obtained with eggs pre-treatedwith nicotine (Text-fig. 65).

464 R. PRESLEY AND P. F. BAKER

Comparison of rates of primary fertilization and re-fertilization

Evidence that re-fertilization of eggs takes place at a lower rate than that of initialfertilization was found from analysis of the distribution of sperm among samples of100 eggs. Text-fig. 7 compares this distribution with that predicted for a Poisson

20 30

Time (sec.)20 30

Time (sec.)

Text-fig. 6. Effect of removal of jelly coat (E. esculentut). (A) Fertilization rate curves asdetermined with KC1. Normal eggs (O); eggs washed free of jelly coat in A.s.w., pH 5-3 andthen resuspended in A.S.W. (•). Sperm density 3 x 10' sperm/ml. Temperature 160 C. Spermadded at zero time. (B) Normal (O); and jelly-free eggs (•); pretreated for 10 min. with12-5 nM nicotine in A.S.w. (pH 8-o). Temperature 16° C. Sperm density 4X io* sperm/ml.Mean sperm per egg plotted against time, as determined by counts on 25 eggs after inactivationwith lauryl sulphate.

100

I 50

0 1I

(Q (D)

0 1 2 3 4100 r

50 -

nii\iiiii

0 1

—

2 3 4

»1t1tI

K

0 1

-

2 3 4

11\tt1

3 1 2 3 4 1

Number of male pronuclel in egg

2 3 4

Text-fig. 7. Comparison of primary fertilization rate with re-fertilization rate (E. esculentus).Histograms of numbers of male pronuclei in eggs from samples of 100. Solid columns indicateexperimental findings; broken lines link horizontals indicating predictions from Poisson functionappropriate to number of eggs in initial condition in each case. Upper row: initial conditionunfertilized; lower row: initial condition monospermy. Vertical columns: (.4) 5 sec., KC1' kill',(B) 15 sec, KC1 'kill', (C) 80 sec., KC1 'kill', (D) 80 sec., UNO, 'kill'. Sperm density5 x ioT sperm/ml. Temperature io° C.

Kinetics of fertilization in the sea urchin 465

knstribution (see Methods section). It can be seen that the number of polyspermiceggs in samples having a large proportion of fertilized eggs is considerably less thanthat predicted by chance from the proportion of unfertilized eggs in the sample. Inthe case of re-fertilization of monospermic eggs the results suggest that there can beno marked decline in the rate of third and subsequent fertilizations over the second.

To exclude the possibility that during the period of fertilization there was a markeddecline in the fertilizing power of the sperm, rate determinations were made on normalunfertilized eggs using either supernatant sperm which had been used to fertilizeanother sample of eggs for 180 sec. previously or sperm which had been exposed for180 sec. to egg-free A.s.w. in which unfertilized eggs had been shaken. Fertilizationrates so obtained were as fast as those obtained with fresh sperm at comparabledensities.

100 n

"5S, 5°

' ' • • ' I I 1 130 60 90 120

Time (iec.)

150

Text-fig. 8. Relationship of fertilization rate curves to development of fertilization membraneand loss of nicotine sensitivity (E. aculentui). Fertilization rate, determined with KC1 (A),and with UNO, (O). Percentage of eggs in a sample of ioo, fixed in formalin at time point,showing initiation (•), and completion (•), of fertilization membrane. Percentage of eggs,in samples of 25, showing four or fewer male pronuclei after addition of nicotine to give a con-centration of 12-5 mM (x) . Sperm density io§ sperm/ml. Temperature 130 C. Sperm addedat zero time.

Determination of the time of elevation of the fertilization membrane

It is of interest to compare the time-relationships of the fertilization rate curveswith the initiation and completion of fertilization-membrane production, because theelevation of the fertilization membrane is generally thought to provide a completeblock to further fertilization.

In the course of our experiments with nicotine it became clear that nicotine canpromote polyspermy if applied either before or just after fertilization (Baker & Presley,1966). The final loss of sensitivity to nicotine seems to correlate well with the time offertilization-membrane elevation and provides a simple method of estimating the timeof appearance of fertilization membranes in a sample of eggs. The duration of nicotinesensitivity in a population of eggs in relation to the cortical response to fertilizationwas investigated by fertilizing samples of eggs under identical conditions and adding

466 R. PRESLEY AND P. F. BAKER

nicotine at various time intervals after fertilization to give a final concentration oft12-5 DIM, mixing being obtained by hand agitation. In each case fertilization pro-ceded for a total of 180 sec, except that where nicotine was added at 180 sec. inactiva-tion was at 240 sec.; in the subsequent scoring (after KC1 inactivation) eggs showingfive or more pronuclei were counted as having been in a nicotine-sensitive state at thetime of addition of nicotine, such eggs not being present in samples where nicotine-free A.s.w. was added with agitation. The time course of the cortical response wasstudied in normal eggs at the same sperm density and temperature by the formaldehydefixation method of Allen & Griffin (1958). Text-fig. 8 shows the time relationship ofthe fertilization curves as determined with KC1, UNO3, the cortical responses, and theduration of the nicotine-sensitive stage.

DISCUSSION

The choice of spermicidal agents is restricted by the need for the eggs to developafter treatment, while the sperm must be prevented from effecting further fertilization.It is not known at what stage in its interaction with the egg a sperm becomes insensitiveto a particular spermicide, and it is quite possible that after transfer to a spermicidethere is a reservoir of sperm close to the egg which can continue to effect fertilizationfor some time. Any error resulting from this will show a greater degree of fertilizationat a given time point than is actually the case, and so a tendency for one agent to givea lower fertilization rate than another may signify its greater efficacy.

Errors in the method of scoring fertilization by cleavage count may arise fromparthenogenetic activation of unfertilized eggs, asynchrony of cleavage in the population,or where polyspermic eggs divide normally or fail to cleave. In the case of the micro-scopic pronuclear count there may be a tendency to err on the low side in scoring, assperm which have entered an egg but lagged in subsequent development may not becounted when the criteria of development are strictly applied. Thus when this methodindicates a high degree of polyspermy, great significance may be attached to thefinding.

All our results have indicated that the rate curves fall into three groups: that asdetermined with 30-A.s.w.; those with KC1, lauryl sulphate and EDTA; and that withUNO3. It is clear that the results obtained with 30-A.s.w. may include a systematicerror due to the lack of immediate efficacy, and also the tendency to promote poly-spermy at short time intervals where high sperm densities are employed. KC1, laurylsulphate and EDTA all act at an earner stage than UNO3, and all agents act at anearlier stage than the visible cortical response and the disappearance of the nicotine-sensitive stage. We have presented evidence elsewhere (Baker & Presley, 1969) thatthe difference between the UNO3 curves and the others is due to UNO3 blockingfertilization at a different stage from the other agents.

With KC1, lauryl sulphate and EDTA our finding of curves of a sigmoid shapeboth in the presence and absence of jelly are in agreement with those of Hagstrom(1956), but not with those of Rothschild & Swann (1952), whose results were obtainedwith dilute sea water as an inactivating agent. An initial 'shock' causing a lag in spermmotility on change of environment might contribute to the shape of the rate curve inthe early stages, as might the necessity for the successful sperm to pass through an

Kinetics of fertilization in the sea urchin 467

lintermediate stage of penetration during a time interval of the order of seconds beforeceasing to be susceptible to the spermicide. It is clear, however, that the removal ofthe bulk of jelly coat does not completely abolish this lag. The presence of a similarlag in the fertilization of nicotine-treated eggs suggests that the mechanism of spermentry in this case is similar to that in normal fertilization.

Rothschild & Swann (1952) used rate curves of similar shape to those obtained byus with 30-A.s.w. to compare the initial rate of fertilization with that of re-fertilizationin P. miliaris. They concluded that, within 3 sec. of fertilization, a rapid change waspropagated round the egg surface, reducing the fertilization rate to ca. 1/20 of theinitial rate. Our results with 30-A.s.w. suggest that a tendency to promote polyspermy,coupled with a lack of immediate efficacy, make for a considerable error in the analysisof rates determined with this agent in E. esculentus', though less so in P. miliaris.Despite this, in both species, our findings on the analysis of sperm distribution are ingeneral agreement with those of Rothschild & Swann that re-fertilization takes placeat a lower rate than initial fertilization. Despite the approximations in the calculationof probability, it is also very clear that in E. esculentus the entry of subsequent spermdoes not markedly reduce the re-fertilization rate below that produced by the firstfertilizing sperm, a finding which is in keeping with the concept of an 'all or none*fast partial block to polyspermy produced by the normal egg in response to the firstfertilization, and which also suggests that no marked decline in the fertilizing powerof the sperm suspension takes place during the time in which the eggs remain sus-ceptible to sperm entry.

It should be noted that these experiments do not establish at what time point in theinteraction between sperm and egg a 'fast blocked' condition of the egg may arise;if it is possibe for UNO3 to block sperm entry as much as 30 sec. after an egg wouldhave appeared fertilized as determined by KCl, the possibility cannot be excluded thatthe mode of exclusion of supernumerary spermatozoa by the egg involves action atsome similarly late point in sperm entry.

SUMMARY

1. A method is described for the direct counting of male pronuclei in recentlyfertilized sea-urchin eggs.

2. Using this method, fertilization rate determinations were made to compare 30%artificial sea water (A.S.W.), isotonic KCl, sea water containing lauryl sulphate, calcium-free and magnesium-free A.s.w. containing EDTA, and sea water containing uranylnitrate, as agents blocking fertilization but permitting further development ofpreviously fertilized eggs.

3. 30% A.s.w. was found to be less satisfactory than the other agents, lackinginstant effect, and tending to promote polyspermy. The other agents all gave sigmoidrate curves, that of uranyl nitrate lagging 15-25 sec. behind the others.

4. Evidence was found that uranyl nitrate acts at a later stage in fertilization than theother agents.

5. Sigmoid rate curves were found, except with 30% A.S.W., when eggs with thebulk of the jelly coat removed, and nicotine-treated eggs, were fertilized.

6. Analysis of sperm distribution among eggs from samples fertilized for more than

468 R. PRESLEY AND P. F. BAKER

40 sec. confirmed that re-fertilization takes place at a lower rate than primarjdfertilization.

7. The processes blocked by KC1 and uranyl nitrate were found to precede thecortical responses to fertilization, and the termination of nicotine sensitivity.

During this work R.P. was in receipt of a grant for technical assistance from theMedical Research Council.

REFERENCES

ALLEN, R. D. & GRIFFIN, J. L. (1958). The time sequence of early events in the fertilization of seaurchin eggs. I. The latent period and the cortical reaction. Expl Cell Res. 15, 163-73.

BAKER, P. F. & PRESLEY, R. (1966). A direct method of measuring the rate of entry of sperm into seaurchin eggs. J. Pkytiol. 186, 47-9P.

BAKER, P. F. & PRESLEY, R. (1969). Kinetic evidence for an intermediate stage in the fertilization of thesea urchin egg. Nature, Lond. aai, 488-90.

CLARK, J. M. (1936). An experimental study of polyspermy. Biol. Bull. mar. biol. Lab., Woods Hole 70,361-84.

HAGSTROM, B. E. (1956). Studies on the fertilization of jelly-free sea urchin eggs. Expl Cell Res. 10, 24-8.HAGSTROM, B. E. & HAGSTROM, B. (1954). A method of determining the fertilization rate in sea urchins.

Expl Cell Res. 6, 479-84.HERTWIG, O. & HERTWIG, R. (1887). Experimentelle Studien am tierischen Ei vor, warhend und nach

der Befruchtung. Jena. Z. Natuno. 20, 120-Z42.OKAZAKJ, R. (1956). On the possible role of high energy phosphate in the cortical change of the sea

urchin egg. Expl Cell. Res. 10, 476-504.ROTHSCHILD, LORD & SWANN, M. M. (1950). The fertilization reaction in the sea urchin egg. The

effect of nicotine. J. exp. Biol. vj, 400-6.ROTHSCHILD, LORD & SWANN, M. M. (1952). The fertilization reaction in the sea urchin. The block

to polyspermy. J. exp. Biol. 29, 469-83.RUNNSTHOM, J. (1961). The mechanism of protection of the egg against polyspermy. Ark. Zool. 13, 565.

EXPLANATION OF PLATE

Echinus esculentus

A, B; bright ground appearance of monospermic (A) and dispermic (B) eggs after development andstaining procedure described in methods section, x 200.C. Phase-contrast micrograph showing male pronucleus in optical section at higher power. Egg fixedand stained according to procedure described, x 1600.D. Bright ground micrograph of egg after similar treatment to A and B showing many pronuclei inoptical section. From 40 sec. sample, KC1 'kill', pretreated for 10 min. with 12-5 mM nicotine. Spermdensity 4 x io7. x 300.E. Dark-ground micrograph of living egg from same conditions as in D taken at time of first cleavagein control eggs, to show regular appearance of highly polyspermic eggs at this stage and only faintvisibility of multiple spindles, x 400.

Journal of Experimental Biology, Vol. 52, No. 2 Plate 1

R. PRESLEY AND P. F. BAKER (Facing p. 468)