Acid and base production at fertilization in the sea urchin

E.rperime&d

Cell Research 22, 233~245 (1961)

233

ACID AND BASE PRODUCTION IN THE J. W. MEHLl Department

of Zoology,

AT FERTILIZATION

SEA URCHIN and M. M. SWAIYN Unicersity

of Edinburgh,

Scotland

Received March 11, 1960

A RURST of acid production n-as shown hi’ Runnstrijm [ll] to be one of the reactions which follows the fertilization of sea urchin eggs. Despite a number of investigations, the nature of this acid has not been determined. It does not appear, however, to be lactic, malic, pyruyic or phosphoric acid [i]. Quite recently Rothschild [Sl has re-examined lactic. acid production following fertilization, and has confirmed the earlier c.onclusion that it cannot account for the “fertilization acid”. The extra acid production has commonly been demonstrated as an increased evolution of CO, in the LVarburg apparatus, but in RunnstrGm’s first esperiments it was shown that the bicarbonate of the medium is depleted, and that the pH calculated from the bicarbonate concentration and the pC0, decreases. In his most recent paper, Rothschild [S] also has shown that the bicarbonate of the medium is depleted during the formation of the “fertilization acid”; while =\llen et al. [2] hare shown that the pH of unbuffered egg suspensions drops during the first few minutes after fertilization. The more obvious metabolic acids which might be produced in increased amounts after fertilization seem to have been eliminated, hut a number of other possibilities remain. There might be a release of sulphuric or hpclrochloric acid, though the amount of sulphate in unfertilized sea urchin eggs is barely sufficient to account for the observed acid production, eren if it were all liberated [Yl. -Uternativelp, as Rothschild [i j has suggested, the acid production may simply be due to an exchange reaction inrolklg hydrogen ions. This might come about in various ways as a result of metabolic reactions. Equally likely, in view of the. obvious changes in the egg following fertilization, would be the exposure of new acid groups on the egg surface, or alterations in the ionization constants of groups already exposed, as a resu!!, perhaps, of some change in the proteins. 1 Work carried out during the tenure of a Fellowship address: Department of Biochemistry and Nutrition.

from the Commonwealth rniversity of Southern Experimrnial

Fund. Permanent California. U.5.A. Cell Research 22

234

J. W. Mehl and Ad. M. Swarm

Lye were interested, first of all, in trying to eliminate the possibility that a weak acid might be released from the egg. \\‘e therefore compared the titration curves of the supernatant solutions from fertilized and unfertilized eggs. Our failure to find any differences of a magnitude sufficient to account for the ‘fertilization acid” led us to consider the possibility of changes in the egg surface. Among the questions investigated in this c.onnection was that of the relation between pH and the amount of “fertilization acid” produced. It was found that changes in pH have a marked effect, and that below about pH 5.3 a “fertilization acid” is no longer formed. Rather, base” is formed. Although the exact acid is taken up or a “fertilization mechanisms involved are by no means clear, it is evident that the phenomenon of acid production can no longer be regarded as being due to the release of some single metabolic product.

METHODS

The sea urchins, Echinus esculentus (Linn.), were obtained from the Gatty Marine Laboratory, St. Andrews, in May and June, and from Millport Marine Biological Station in late June. Psammechinus miliaris (P. L. S. Muller) and heart urchins, Echinocardium cordatum (Pennant), were obtained from Millport in late June. The experiments were done in part at St. Andrews and in part at Edinburgh. Spawning of females was induced with 0.5 AI KC1 solution, and in all cases the jelly coat was removed from the eggs by the method of Hagstriim [4]. Jelly can readily be removed from the eggs of Echinus and Psammechinus by brief treatment with sea water at pH 5.0. The eggs of Echinocardium require rather longer treatment at pH 4.5. The volume of unfertilized eggs was measured by centrifuging an aliquot of the dilute suspension in a 3 ml milk sediment centrifuge tube. Calculated volumes of eggs could then be obtained by taking a proper volume of this dilute egg suspension, which was kept very gently stirred by a slowly rotating paddle. The volume of eggs always refers to the volume occupied by the jelly-free unfertilized eggs. As the eggs were required, they were washed repeatedly with bicarbonate-free sea water by allowing them to settle, and were finally suspended in the desired volume of bicarbonate-free sea water. Semen was obtained by cutting up the gonads, and was used without dilution to give a final concentration of about 4 x 10’ spermatozoa/ml. It can therefore be assumed that virtually all the eggs are fertilized within a few seconds of adding the sperm [lo]. Bicarbonate-free sea water was prepared by adjusting sea water to pH 3.0, and boiling to remove the CO,. After cooling, the pH was readjusted to 8.0 or to that desired for the particular experiment. pH measurements were made with a Pge Model 11067 meter and Nos. 11126 and 11161 electrodes. In order to keep the volume of solution at a minimum, the automatic temperature compensator was not used. Experimental

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;Icid und base production at fertilization

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IiOH used for the titrations was standardized as an approximately 0.2 N solution with analytical potassium acid phthalate. Additions of IiOH or HCl were made with a syringe-driven borette. RESULTS ,4cirJ production at pH S.O.-A somewhat different method from those previously employed, was used to measure the amount of the fertilization acid formed. The egg suspension, 15 to 20 per cent by s-olume in concentration, was placer1 in a beaker containing the electrodes of the pH meter and a slow-speed stirrer. The pH was adjusted to 8.0 with KOH. Sperm was introduced at zero time, and KOH xv-as then added as required to maintain the pH at 8.0, the amount added being noted at appropriate times. The results of three such experiments with Echinus esculenfus are seen in Fig. 1. The amount of base added up to a given time is shown as /(moles of acid produced per ml of eggs. Xn increase in acid production takes place, beginning at 1.5 to 2 minutes after fertilization and continuing for 2 to 3 minutes after l-his. Subsequently, acid production becomes essentially linear for aa least the nest ten minutes. \\‘e have assumed that this linear portion extrapolated to zero time gives the extra acid, or “fertilization acid”. The three experiments are in good agreement with respect to the time

TIME-MINUTES

Fig. l.--Acid production following fertilization of eggs of Echinus esculerztus. Eggs were fertilized at pH 8. at 0 time, and this pH was maintained by measured additions of KOH. The KOH required has been expressed as pmoles of acid produced per ml of eggs. The dashed lines are estrapolations of the linear portion, which is taken to represent the part of the acid production due to CO,. Ercperimental

Celf Reseurch 22

J. IV. Mehl und M. M. Swarm

236

relationships, although the batch of eggs studied on May 12 gave about 18 Both the time course and the quantity of per cent greater acid production. acid are similar to those previously reported.

0.‘t,.( I

2

3

4

5

6

7

B

9

TIME-MINUTES

of eggs of Psammechinus milioris which had been Fig. Z.--Acid production followin, 0 fertilization pre-treated with trypsin. Experimental conditions as for Fig. 1, but fertilization membranes were not formed in this case as a result of the trypsin treatment.

Identical experiments were also done with Psammechinus miliaris, and gaT-e very similar results (see Fig. 4), though the sequenc.e of events is somewhat more rapid. Acid production begins at about 1 min after fertilization, and is virtually complete within a further 2 mins. Similar results again were obtained in a single experiment with Echinocarclium corclatum. An experiment was also done to test the effect of digesting away the ritelline membrane of the unfertilized egg with trppsin, so preventing the elevation of fertilization membranes. It will be seen from Fig. 2 that acid production is unaffected. Titration of supernatant sol&ions.-The fertilized eggs were removed by low-speed centrifugation, and the resulting supernatant was cleared of sperm by centrifuging at high speed. An identical volume of eggs for the unfertilized control n-as washed and suspended in bicarbonate-free sea water, and then stirred for the same period as the fertilized eggs. In two experiments with Echinus esczzlentus, measured volumes of the supernatants were titrated by adding HCl from the microburette, and measuring the pH after each addition. These titrations also include the bicarbonate formed by the eggs during the experiment. In the experiment with Psammechinus miliaris, suffmient HCl was added to reduce the pH to about 2.3. The solution was evacuated to remove C02, and the titration was then done with KOH. These results are shown in Fig. 3, and are plotted as pmoles of Experimental

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=Icid and base production at fertilization

237

hydrogen ion bound or released per ml of eggs. The curves have all been adjusted to zero binding at pH 5.3, since this seemed to be a region in n-hi&h binding was changing little with pH. The bar given with each curve represents the amomlt of fertilization acid formed in that particular esperiment, and

P MOLES

H+

BOUND

PER

ML

EGGS

Fig. 3.-Titration of supernatant solutions from fertilized and control eggs. (a) Echinus esculentns, (h) Pscmmechinm miliaris. In both cases the eggs were kept at pH S until release of “fertilization acid” was completed, or the same length of time for the control. In (a) titration was begun at pH 8 and carried out with HCl. In (b) the supernat.ant was first adjusted to about pH 2.3 and the CO, removed, and the titration was then carried out Mth KOH. Binding of Hf is expressed as pmoles per ml of eggs in the suspension from which the supernatant was taken, and the amount of fertilization acid formed in each case is shown by the corresponding bar.

it will be evident that there is no region in which the curve for the fertilized supernatant differs by this amount from the unfertilized. There appears to be a difierence of about 2 /“moles per ml of eggs in the region of pH 2.5 in the experiment with Psazzzmechinrzs zniliczris, but this is in the wrong direction, and is probably due to problems that were encountered with drifting of the elec,trode in more acid solutions. The results below pH 3 are not very reliable, but it is quite clear from all three experiments that there is no acid released Esperimental

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J. 11’. Mehl and M. hf. Swam

238

into the supernatant with a pK between 3 and 8 in sufficient quantity to account for the “fertilization acid”. Fertilization acid us a function of PH.--If the burst of acid production were due to changes in acid strength of groups already in the egg surface or to the appearance of new weakly acidic groups, the amount of acid produced might depend upon the pH of the medium in which the measurements

4 3 2 l/-c,

0

-.-* 2

3

4

5

:

,

,

,

6

7

B

9

pH

7 TIME-

.-

PH 7-o

9.0

I

9

MINUTES

Fig. 4.-Acid production following fertilization of eggs at three pH values. Psammechinus miliaris. Egg suspensions mere adjustecl to the indicated pH, fertilized at 0 time, and the amount of KOH required to maintain the pH at this values was recorded at subsequent times. The amount of KOH required has been expressed as ,umoles of acid produced per ml of eggs. The dashed lines represent the rate of CO, production, which is superimposed upon the production of fertilization acid. Experimental

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A4cid and base production at fertikation

239

were made. This would be particularly true if the pH were near the pK of the groups involved. The pH might also, of course, influence the amount of a metabolic acid produced, but it was thought that the nature of any changes which were found might provide some basis for an improved understanding of the phenomenon.

4.81 cl

2

4

6 TIME-

8

10

12

I 14

MINUTES

Fig. S.-Production of base at pH 4.93, following fertilization of eggs of Echinus escuienfis. Eggs were fertilized at pH 7.6 at 0 time. Acid was added at 1 min 15 see to reduce pH. Stabilized at pH 4.93 at 2 min 15 sec. The pH subsequently rises rapidly in the period in which “fertilizatiox acid” is formed at higher pH values.

_%series of experiments were therefore made as before, but with the initial pH adjusted to some value other than S.O. Between pH i.0 and 9.4 the sperm were added at the pH at which the experiment n-as done. &It the lower pH values, fertilization was carried out between pH 7.0 and 8.0, 0.5 to 1 mins were allowed for fertilization to take place, and the pH was then reduced ad rapidly as possible. Fertilization membranes were x-cry imperfect or did not form at the lower pH values, but fertilization had evidently taken place as judged by the appearance of sperm asters and subsequent cleavage furrows. Three experiments, at pH 7.0, 8.0 and 9.0 with Psmnmecl~irws milimis, are shown in Fig. 4, Production of “fertilization acid” clearly increases with increasing pH, as does CO, production. Since the effect becomes smaller at lower pH values, it is most clearly seen when the changes in pH are recorded. One such experiment with Pstrn7ntechinru eggs is shown in Fig. 5. The eggs were fertilized. at pH 7.6 and acid was added about 1 min later. The pH had stabilized at 4.93 by 2 mins. Between 2 and 4 mins the pH rose rapidly, and then became relatively constant after . 3 mms. Xfter S min, 0.7 k&moles of HCl were required per ml of eggs to return the pH to 4.93. The time relations were the same as those observed in the more alkaline media, but the pH change was in the opposite direction. The results of a number of experiments are given in Fig. 6. -Though acid” formed per ml of eggs, it is clear presented as j.tmoles of “fertilization Experimentnl

Cell Resenrch 22

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J. Ty. Mehl and M. M. Swann

for both Psammechinus and Echinus that acid is not formed below pH 5.2-5.3, but that there is production of base. This sort of behaviour c,ould result from a number of mechanisms, but it is worth noting that there is no indication of a discontinuity in the curve. Indeed, both curves seem to be very nearly linear throughout the entire region studied. This seems to us to suggest that results obtained at pH 5.3 and below are signitkant in a general way for the process in question.

Fig. 6.-Fertilization acid as a function of pH. Calculated from the total amount of acid or base required to maintain constant pH after fertilization, but corrected for the constant increase due l , Echinczs esczzlentzzs, 0, Psammechinrzs miliaris. to CO, production.

The observed behariour could be due to the release of both strong acid and base following fertilisation, the relative amounts of the two being dependent upon the pH of the medium. It could also result from a change in the pKs of acid and basic groups in the egg surface, or from an increase in the number of such groups. Since the egg surface certainly c,ontains proteins which are likely to undergo substantial changes associated with the obvious physical c.hanges in the egg surface, the second posibility seems particularly attractive. Titration of fertilized and unfertilized eggs.-The possibility that ionizing groups in the cell surface are changing in quantity or in strength can be Experimental

Cell Research 22

Acid and base production TABLE

241

at fertilization

I. The change in acid or base-binding capacity of eggs (Psamnechinus miliaris) following fertilization. Experiment a b c

d

I

1 ml of eggs in 5 ml bicarbonate-free waler were adjusted to pH i. The pH was raised to 9.0. Fertilized at 0 time and maintained 9.13 for 9 minutes

Returned pH of suspension 10.5 minutes.

sea

Required 9.25 {u&es KOH. the pH at Required 13.0 pmoIes KOH of which 7.6 pmoles fat- fertilirafion acid and S.4 pmoles for CO,.a to 7 between 9 and Required 13.1 /
Calculation e

f 9

Difference between fertilized and unfertilized 3.8 pmoles per ml eggs. eggs (d - b). Correction for titration of CO, between pH 9 and 7 (from cb). 2.5 pmoles. Corrected difference between fertilized and 1.3 !lmoies per ml eggs. unfertilized eggs (e - f).

Erperimenl a b c d e f

I?

2 ml eggs in 10 ml bicarbonate-free water were adjusted to pH i. Reduced pH to 5.3. Returned pH to 7.0. Fertilized at 0 time and added 1.28 at 40 sec. Initial pH, 5.20. At 6 minutes, pH had risen to 5.39. pH to 5.20. Returned pH to 7.0.

sea Required 1.2X pmoles HCljml egggs. Required 1.11 pmoles KOH/ml eggs. pmoles HCl per ml eggs Returned Required 0.37 pmoies HCljml eggs. Required 4.06 ;imoles KOH/ml eggs.

Calculation Y h i

Difference between fertilized and unfertilized 2.75 pmoles per ml eggs. eggs (f - average of h and c). Correction for titration of CO, between pH 5.2 and 7.3.’ 0.1 pmoles per ml eggs. Corrected difference between fertilized and unfertilized eggs (g - h). 2.35 ,umoles per ml eggs.

a See Fig. 4. b Taking pK, as 6.03 and pK, as 9.0, and assuming that CO, production has continued at the rate indicated at pH 9 for the entire 10.5 minutes. ’ On the basis of the rate of CO, production at pH 7 (Fig. 1). This would represent a maximum or indeed, an overestimate. 16 - 60173258

Experimental

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J. IV. MehI and M. M. Swann

investigated, at least in principle, by direct titration of living fertilized and unfertilized eggs. Two exploratory experiments were in fact carried out, but it was apparent that a number of fac.tors contribute to making the errors rather large. The uptake of hydrogen ions by the eggs is remarkably small, of the order of 10 to 20 F&moles per ml of eggs between pH 9 and -I; but when the titration is done serially, on one batch of eggs, there is no very satisfactory way of correcting for respiratory CO,. Other difficulties are involved in measuring the volume of eggs and solvent with sufIic.ient accuracy, and in eliminating errors due to cytolysis, which leads to a large increase in acid and base binding. These difficulties may to some extent be avoided by comparing the amount of acid or base required to titrate a single batch of eggs between two pH values before and after fertilization. Two such experiments were done. Although giving somewhat equivocal results, they both point to the same conclusion, and seem worth reporting. In Exp. 1 of Table I, a batch of 1 ml of Psammechinus miliaris eggs was adjusted to pH 7. The amount of KOH required to raise the pH to 9 n-as measured, and the eggs were then fertilized at pH 9. The acid evolution was measured for the next 9 mins in order to obtain the “fertilization acid”and the rate of CO, production. The eggs were then titrated back to pH 7. After correcting for CO, produced, it could be calculated that 1 .i !&moles more base is required to titrate fertilized eggs between pH 7 and 9 than are required for unfertilized eggs. Although the uncertainty in this value is of the order of 0.4 F&moles, there is clearly a difference in the titration of fertilized and unfertilized eggs. However, the difference in acid production between pH 7 and pH 9, estimated from other experiments (Fig. 6), is obviously larger (around 5 [[moles). In Exp. 2, a similar experiment was made between pH 5.3 and 7. These are somewhat more favourable conditions with respect to the magnitude of the c.orrection for CO, production. In this c.ase, the amount of acid required to reduce the pH of the eggs from 7 to 5.3, was first determined. The eggs were then returned to pH 7, the amount of alkali giving a second value for the titration of the unfertilized eggs. The eba 0”s were immediately fertilized at pH 7, and after 45 seconds were again acidified. The pH changes were then recorded for G minutes from the time of fertilization, the amount of acid required to return the eggs to pH 5.3 was measured, and the eggs were finally titrated to pH 7 again. In this case, the total difference between the titration of fertilized and unfertilized eggs was 2.75 /[moles per ml. The correction for CO, could not be larger than that at pH 7, which would be 0.4 /&moles per ml of eggs from Fig. 4, and probably would be appreciably less. There is E.rperimental

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Acid and base production at fertilization

243

clearly a difference between the acid-binding capacity of the fertilized and unfertilized eggs. However, the value of about 2.35 !l,moles per ml of eggs is again smaller than the difference between acid production at pH 7 and that at pH 5.3, approximately 3.5 Frmoles from Fig. 6. DISCUSSION The titration of egg supernatants makes it quite clear that there is no weak acid with a pK between 3 and S released into the supernatant after fertilization in an amount greater than about 0.5 Llmoles per ml of eggs. The number of organic, acids with pKs below 3 is limited. =tlternatirely strong acids such as hydrochloric or sulphuric might be released, though, as MY eggs is barely pointed out earlier, the amount of sulphate in unfertilized sufficient to account for the acid produced. !KTe come nest to our findings that the amount of “fertilization acid” is strongly dependent on the pH of the medium, and that a part of this dependence can be explained by a change in the titration of the egg after fertilization. These facts leaye little doubt that at least half the acid production is due to changes in the strength or quantity of ionizing 3oroups that are attached to the eg.g. Tf n-e take pH 5.3 at which there is no acid or base formation, as the point of reference, an apparent acid production of about 4 pmoles per ml of Pmrnrnechinus miliaris eggs can be espected at pH 9 as the result of the change in the titration curve. This, however, would account for only part of the total observed. An additional acid production of approximately 3.6 E&moles per ml of eggs must be due either to the formation of a strong acid, which is not erident i’n the titration of the supernatant solution from the fertilized eggs, or to the appearance of new groups on the egg surface with pKs substantially below pH 5.3. \Vhatever the source of this stronger acid, the amount formed must he dependent upon the pH of the medium, or increasing amounts of a relatively strong base must be formed as the pH decreases in order for acid formation to become zero at pH 5.3. It might be possible to choose between a change in groups on the surface of the egg and the formation of a diffusible stron, 0 acid, with the aid of studies of the surface potential of fertilized and unfertilized eggs. Afeasuretnents of this kind hare in fact been made on eggs of drbacia pmriulnfn bp Dan [3:, but they do not indicate changes of any considerable magnitude in fbe surface charge as a consequence of fertilization. It is evident, hoverer, that the measurement of surface potentials of marine eggs presents serious problems, because of their large size.

We have so far assumed that the groups responsible for the appearance of fertilization acid (or base) are situated on the egg surface, but there are If the groups in question mere certain difficulties about this assumption. on the outermost surface of the egg, there would onl? be an area of about 1 KZ for each group. If, on the other hand, we suppose that the groups are arranged in depth, the situation becomes more reasonable. Assuming that the ionizing groups belong to amino acids, and that one amino acid in twenty gives rise to a completely ionized group, then a layer of protein (hydrated to the same extent as the rest of the egg) of about 1.5 p could account for the observed acid production. The cortex of the sea urchin egg has in fact been estimated to be about 1.5 J&thick [S], but it is normally supposed to lie inside the per~neabili~~ InembraIle. It is, of course, possible that the hydrogen ions escape awoss the pernleabili~ barrier, but if so the ionic flux must be of the order of 100 ,umoleslsq.cm/sec. This figure is very much greater than those recorded for all other cases (e.g. nerve cells, Nitda etc.) where ion movement has been measured. It is indeed so large that one would be forced to conclude that the permeability membrane must have broken down completely. Unlikely as this may seem, it might account for the well-known fact that the sea urchin egg, though quite resistant at other times, is extremely easily cytolysed in the first few minutes after fert~izatio~~ [5]. Certainly the cortical granules escape through the permeability layer if this is at the egg surface, and it is possible that part of the observed effect might be due to their coming into a position where they can react freely with the environment. On the other hand, there is evidence, reviewed by Allen [l], that suggests that th.e cortex may be outside the permeability membrane. The question therefore cannot be regarded as settled, but we incline to the view that the acid production due to groups in the egg is in fact the result of changes in material at or barrier, at least after near the egg surface, and lying outside the permeability fertilization. Three main points arise from this work. It is clear that no weak acid is released into the supernatant at fertilization, and that at least half and perhaps the whole of the acid production is due to the appearance in or on the egg of new acidic groups which then release hydrogen ions into the medium. In addition there is good evidence that basic groups also are exposed. SUMMARY

1. Acid production at fertilization in three species of sea urchin egg has been measured by titrating the acid with base as it is formed. -4cid production begins at 1-2 minutes after fertitization and is complete by 4-5 minutes.

2El

A4cid and base production at fertilization

2. ?I comparison of titration curves for the supernatant solutions from unfertilized and just fertilized eggs, shows no significant differences. It is clear that no acid is released into the supernatant with a pI< between 3 and 8, in sufficient quantity to account for the “fertilization acid”. 5’. The amount of acid production n-as determined at pH’s ahore and below that of sea water. dt pH 9.0 it is increased, at pH 7.0, it is reduced, and at pH 5.3 it is nil. Below pH 5.3 there is a corresponding evolution of base. 4. This pH dependenc,e of acid production suggests an ion-exchange 00 and the surrounding sea water. reaction between groups attached to the ebb Experiments in which living eggs were titrated from one pH value to another, both before and after fertilization, show that at least half the acid production is in fact so caused. The remainder may he due to release of strong acid or base, in proportions depending on pH, or to further groups attached to the eqg, with pIls well below 5.3. I 5. It is concluded that acid production is most probably due to changes in the number or strength of acidic and basic ionizing groups attached to the egg, resulting from changes in the state of certain egg proteins. This material may be on the outermost surface of the egg, outside the permeability membrane. If, on the other hand, it is inside the egg, the very high ionic flus suggests that the permeability membrane must break don-n completely for a few minutes after fertilization. We are indebted to the Directors and Staffs of the Marine Biological Station, Millport, and the Gatty Marine Laboratory, St. Andrews, for their kindness and help. REFERENCES

R. D., A Symposium on the Chemical Basis of Development, pi 17. The Press, Baltimore ~1955. 2. ALLEN, R. D.. MARI~HAM. B. and ROWE, E. C., Exptl. Cell Research 15, 346 3. DAN, I<., J. CeUular Comp. Pkysiol. 3! 477 (1933). I.

ALLEN,

Johns

Hopkins

(1958).

4. HAGSTRBM, B. E., Exptl. Cell Research 10, 24 (1956). 5. HERLANT, RI.. Arch. Biol. (Paris) 30, 517 (1920). 6. MIITCHISON, J. M., Quarf. J. Microscop. Sci. 97, 109 (1956).

7. ROTHSCHILD,

8. ~

LORD,

J. Expfl.

9. ROTHSCHILD. 10. ROTHSCHILD, 11. RUNNSTR~~K

Fertilization.

Methuen & Co. Ltd., London, 1956.

Biol. 35, 843 (1958). LORD and BARNES, H., ibid. 30, 534 (1953). LORD and SWANX, M. RI., ibid. 28, 403 (1951). J.? Biockem. Z. 258, 257 (1933).

ADDENDUM Since

writing

Z. pkysiol. nia,

and

this

paper,

our attention has been who demonstrated base. at fertilization.

Chem. 271, 1 (1941)),

of an unidentified

drawn to the work of ijrstriim (Hoppe-Seyler the production of small amounts of ammo-

Erperimenlal

Celi Research 22