INHIBITION O F FERTILIZATION IN EGGS O F MARINE ANIMALS BY MEANS O F ACID

ALBERT TYLER AND BRADLEY T. SCHEER W'illiam G. Eerckhof Laboratories of Biological SCiences,

California Inatitute of Technology, Pasadena

ONB PLATE (TWEtVE FIQU&ES)

INTRODUCTION

It has been shown (Tyler and Schultz, '32) that fertilization in Urechis eggs is more susceptible to lowering of the pH of the sea water than are the later stages of development. Thus sea water acidified to pH 7.2 is sufficient to prevent fertiliza- tion, whereas if fertilized eggs are placed in a solution as acid as pH 6.4 normal cleavage and development is obtained. I t was further demonstrated that the initial stages of the fertilization reaction, namely, the establishment of the block to polyspermy and the changes in shape of the egg, could be reversed by means of acid sea water. It seemed desirable to repeat and extend these experiments with eggs of other marine animals, and this has been done at various times at several different marine stations. The eggs investigated were those of Paracentrotus lividus, Sabellaria alveolata, Ascidiella scabra and Leptonereis glauoa at Roscoff; Dentalium entalis, Phallusia mammilata, Ciona intestinalis at Naples ; and Den- draster excentricus, Lytechinus anamesus and Strongylocen- trotus purpuratus at Corona del Mar.l 'Part of this work was done at the Station Biologique in %scoff and at the

Stazione Zoologica in Naples. The work at the latter station was done during tenure of a National Research Council fellowship by the senior author. The senior author is indebted to director Ch. Perez and to Dr. G. Tessier of the ROSCOff station md to director R. Dohrn and Dr. €3. Ranei of the Naples station for the courtesies extended him.

179

THE JOUBNAX, OF EXPERIMENTAL Z O h W Y , YOL. 75, NO, 2 FEBRUARY, 1937

180 ALBERT TYLER AEiD BRADLEY T. SCHEER

The first point mentioned above was readily demonstrated to hold for all the eggs investigated; that is, the fertilized eggs developed normally in acid sea water of lower pH than is necessary to inhibit fertilization. In regard to the second point these forms appear to differ from Urechis in their be- havior, but before presenting the data it is necessary to point out certain difficulties that are involved. In the first place the demonstration of reversal of fertilization in Urechis is simplified by the fact that one essential part of the fertiliza- tion process, namely, the penetration of the sperm, is not reversed. The entering sperm is not shoved out again as a result of the acid sea water treatment, but remains inert just below the surface of the egg. Upon re-insemination in normal sea water another spermatozoon enters and both then take part in deveIopment. The first division of such eggs is thus of the typical polyspermic type, and this fact together with the proper control experiments and observations d o r d s a rather simple demonstration that one important part of the fertilization reaction, namely, the establishment of the block to polyspermy, is reversed by the treatment. The reversal of the change in shape could also be easily demonstrated in Urechis. In most of the other forms the penetration of the sperm is a much more rapid occurrence than in Urechis and also the first change in shape observed after fertilization is the elevation of the fertilization membrane. From the experi- ments on Urechis it was evident that there was little possi- bility of effecting a reversal after membrane elevation started. When, as happens in the present experiments, eggs trans- ferred to acid sea water within a short time after insemination fail to develop, it can be assumed 1) that the sperm enters and the egg is blocked in its development, or 2) that the sperm enters and is thrown out again, or 3) that the sperm and eggs fail to unite before the action of the acid sea water has affected them. The first alternative can be readily tested, but it is somewhat dil3cult to decide between the latter two, Since the results reported here differ from those with Urechis in that no polyspermk cleavage occurs upon re-insemination of

ACID INHIBITION OF FERTILIZATION 181

eggs that were treated with acid sea water shortly after fertilization, it leaves the question undecided between the latter two alternatives ; one of which means a reversal of both the penetration of the sperm and the block to polyspermy, while the other means no reversal at all but an effect on the germ cells prior to their union. The possibility of testing these two alternatives will be discussed later. I t will be evi- dent, however, from what has been said, that it cannot be concluded that the eggs reported upon here differ essentially in regard to reversibility of fertilization from Urechis eggs.

It was noted in the experiments with the echinoderm eggs that membrane formation was interfered with after certain treatments with the acid sea water, but that the eggs would nevertheless divide and develop. Two kinds of interference have been observed. In one apparently no membrane is formed and in the other a 'tight' membrane is formed. Both types of behavior have been previously reported by Just ( '29 a) and Moore ( '32). The latter type of phenomenon is of particular interest because of the striking modification of the early stages of development that such eggs undergo. The conditions under which such eggs are produced and the kind of development that they exhibit were therefore investigated in some detail. Some observations were also made on the effect of lowering of the pH of the sea water on the rate of development.

CONCENTRATION OF ACID IN WHICH FERTILIZATION Ia INHIBITED

The inhibiting concentrations of acid in sea water were determined by placing unfertilized eggs in a set of dishes containing a graded series of concentrations from those too weak to prevent fertilization to those stronger than necessary to block the reaction. The eggs were usually inseminated about 5 minutes after being placed in the solution. The amount of sperm suspension employed was small enough to produce no significant change in the concentration of the solution. The usual procedure was to prepare a stock solu- tion containing about 2.0 cc. of 1.0 n HCl in 1000 cc. of sea

182 ALBERT TYLER AND BRADLEY T. SCHEER

Paraoentrotue Sabellaria

Asoidiells C i O l l 8 Phalluaia

Chaetopterna

L ytechinus Ureohie Arbacia Aeteriaa Chaetopterus

Leptonereis

Dentaliurn

Dendraster

water and to make up the series by dilution with ordinary sea water. In some experiments the CO, was removed from the sea water by acidifying to about pH 4 and bubbling air through for 24 hours. The inhibiting concentrations were then prepared by the addition of phosphate and (20,-free NaOH. However, no particular effect of the CO, was noted, the solutions prepared by the first method giving inhibition at the same pH as by the second method. The results of all the inhibition experiments are summarized in table 1. The

TABLE 1

Concentration of acid sea water in which jertiliBatatlon i s inhibited. Stock solutatlon=d.O cc. of 1.0 n HCl in 100 cc. sea water

PH PER CENT OF 6 M C K SOLUTION

50-60% 7.4-7.2 4040% 7.5-7.4

<50% > 7.4 <SO% > 7.4

40-80% 7.5-6.8 50-70 70 7.4-7.0

60-70% 7.2-7.0

50-70% 7.4-7.0 . . . . . . . . . . . . . . . . . . . . .

60-7070 7.2-7.0

60-70% 7.2-7.0

7.2-7.1 (Tyler and Sohultz) . . . . . . . ;:: } (Smith and Clowes)

7.1

ACID INHIBITION OF FERTILIZATION 183

limit in which no fertilization is obtained, and the upper limit at which fertilization drops below the control value. The pH values are for the most part interpolated from determinations by means of the glass electrode on dilutions of the stock solution.

Due to the variability encountered the data are only ap- proximate. Nevertheless, it is evident that the range of pH in which fertilization is inhibited is very similar for the eggs of the various forms investigated. From these data it cannot be concluded that significant differences exist. The data for Urechis caupo (Tyler and Schultz, '32), for Arbacia punctu- lata, Chaetopterus pergamentaceoua and Asterias forbesii (Smith and Clowes, '24c) are also presented in table 1. Smith and Clowes conclude from their data that specific differ- ences in inhibiting concentrations exist in the different forms investigated.

DEVELOPMENT O F EGGS I N ACID SEA WATER SOLUTIONS STRONG ENOUGH TO INHIBIT FERTILIZATION

I t was shown for Urechis that eggs, transferred to an acid sea water solution stronger than necessary to block fertiliza- tion, would develop normally if the transfer were made later than 3 minutes after fertilization. The same result is ob- tained with the eggs of the various forms used in this work with the difference that in most of these the time after ferti- lization at which the transfer to the acid sea water may be made is much shorter.

The results are presented in table 2. It will be seen that in most cases fertilization is prevented if the eggs are placed in the acid sea water less than 1 minute after insemination. For Leptonereis the limit is between 60 and 90 seconds, although a small per cent of fertilized eggs in this case was. obtained at 30 seconds. The eggs transferred after that time developed into normal embryos of at least 1 day old while in the solution. The rate of development is retarded, however, in the acid sea water as will be shown later.

184 ALBERT TYLER AND BFtADLEY T. SCHEER

PEE OENT FERTILIZED

RE-INSEMINATION OF EGGS IN WHIUH FERTILIZATION IS BLOCKED BY ACID SEA WATER

In the Urechis experiments the eggs in which fertilization had been blocked by transfer to acid sea water within 3 min- utes after fertilization would, upon return to normal sea water, resume development if the sojourn in the acid solution

~~$~~~ EMBRYOS

TABLE 2

Eggs transferred to mid aea water after insemination. Stock solution = 8.0 cc. of 1.0 n HCl in 1000 cc. 8ea water. Temperatwe = 30 to W'C.

30 60 90 120 15 30

Paracentrotus

5 5 0.5 0.5 95 95 100 100 1 1 99 99

-

Sabellaria

Leptonereis

0 40 100 0 95 0 10 60 75 85 4 25 75 90 95

Ascidiella

0 40 100 0 60 0 1 5 30 10

Ciona

Phallusia

Dentaliurn

Lytechinus

00NOENTBbTION A6 PE& OENT OF BTOOK SOLUTION

I N BEA WATIR

75

0 (control) 50

50

50

100

80

100 -

0 (control)

100

0 (control)

TI'ME OF TRANSFER TO BOLUTION I N BEOONDB AFTER INBEYINATION

15 30 45 60

15 30 60

600

0 0

30 60 60 0 5 40 90

45 60 90 0

300 0 30 60 90

15 30 120 300

ACID INHIBITION OF FERTILIZATION 185

OONOENTEA- TION AS PEB ENF OP STOOK

SOLUTION I.

was not longer than 10 minutes. Eggs left in the solution for a longer time had to be re-inseminated in order to develop, and then they would develop as polyspermk eggs. They could, however, be made to develop normally by artificially activating them with a treatment that gives no cleavage but only polar body extrusion (Tyler, '31). With the present material the results are somewhat different. As with Urechis, the proper concentration of acid sea water stops the eggs if

TIME O P TBANSPEB TO SOLU-

TION

TAELE 3

Re-insemination of eggs in which f e d i s a t i o n has been blockea by meam of acid sea water. Stock eolution=d.O cc. of 1.0 n HGI in 1000 cc. sea water.

Temperature = $0 to 22%'. Time is given from insemination in all wses

~orrnal

100 0.5 0

95 0 0

95 1 ... ... ...

-- ,,Ezic --

0 0 0 0 0

0 0

--

--

-- .. .. ..

Ciona

100 45 Phallusia 1 $ I 45

45 50 15 Ascidiella

TIME O F EETUBN TO SEA WATEE

mdnutes 24 24

Not 50 50

Not 90

Not 15 15

Not

TIMR OF BE-INSaM- INATION

mdinutee 25

Xot Not 51

Not Not 95

Not 16

Not Not

PEE OENT N O E X U

EMBEYO8

--

95 0 0

95 1

80 95

0

they are transferred to the solution within a short time after insemination. This is illustrated in table 2. Also, as was the case for Urechis, the eggs resume development if returned to normal sea water after a short time and fail to resume development if left longer in the acid. However, the latter, upon re-insemination, develop as normal monospermic eggs, not as polyspermic ones. Some of the results are presented in table 3. In Ciona, for example, eggs transferred to the acid solution at 45 seconds after insemination gave no de- velopment. When a sample of the eggs was returned to

186 ALBERT TYLER AND BRADLEY T. SCHEER

normal sea water at 24 minutes, 0.5% of normal cleavage was later obtained, and from a sample removed at that same time and re-inseminated 100% cleaved normally. In the case of Paracentrotus, listed in the table, development occurred with- out re-insemination when the eggs were returned to sea water at 15 minutes.

The failure to obtain polyspermic cleavage in these forms can be interpreted in two ways, as was pointed out above. The initial effect of the acid sea water may have been to pre- vent the union of the egg and sperm, or to effect a complete reversal of the fertilization reaction involving the expulsion of the entering spermatozoon. The first alternative implies that the actual union of the egg and sperm does not occur within the time limit in which the acid block is effective. It is evident from observation, however, that actual contact be- tween egg and sperm is made within 5 to 10 seconds after insemination. This would mean, then, that in the time be- tween contact and upper limit of acid block the fertilization reaction has not actually started. In other words, the block to polyspermy is not established immediately after the first spermatozoon hits the egg, but some time later, and that all the sperm in contact with the egg start to enter. This seems rather unlikely but in the absence of further information we cannot entirely reject this alternative. The other alternative means that the block to polyspermy is set up immediately after contact of the first sperm. The acid sea water reverses this block and in addition causes the egg to expel the entering sperm. The resumption of development of eggs, returned to normal sea water after a short sojourn in acid, is explained then as re-fertilization by the same or other spermatozoa surrounding the egg. The failure to develop after a longer sojourn in acid means that such treatment renders the sur- rounding sperm incapable of fertilization. The eggs them- selves are not injured by the longer treatment since they develop normally upon re-insemination.

I t has often been noted (Smith and Clowes, '24) that eggs inseminated in concentrations of acid sea water, close to the

ACID INHIBITION O F FERTILIZATION 187

100 75 50 0

inhibiting value, give a high per cent of polyspermy. This is to be expected on the basis of the second interpretation offered above, since if a certain concentration of acid will reverse the block to polyspermy a somewhat weaker concen- tration should retard the establishment of this block. The recent results of Clark ( ’36) in which polyspermy is produced by treatment with various agents including acid sea water may be similarly interpreted.

minutes rnhutee house 71 107 .. 67 91 19 59 88 15 55 80 14

RETARDATION O F DEVELOPMENT I N ACID SEA WATER

Numerous observations, since Loeb (1898), have shown that acidifying the sea water retards the rate of development of

TABLE 4

solution = 2.0 cc. of 1.0 n. HCl in. 1000 co. aea water; temperature = 20.0”;

5 minutes after imemination I I I

Retardation of development of Dedraster eggs in aoid aea water; stock

time is given from insemination. Eggs placed in the solutions

smoM) T I Y E OP ~ASTX.UIATIOI TAIL OP FIBST CONCLNTIUTIOW AS PEB CENT OP STOCK SOLUTION C m V A Q m QIJUVAQS

marine eggs. It is of interest to know if development after fertilization can be reversibly stopped by means of acid. This would have considerable value for studies on the energetics of development that are now in progress (Tyler, ’33-’36). VIBs, Dragoiu and Rose (’23) have determined the pH of sea water necessary to inhibit cleavage, and use this in their notion of ‘travail d’arr6t.’ It was not shown, however, that normal development would ensue after prolonged inhibition of cleavage.

In table 4 data on the retardation of development are pre- sented. It will be seen that the time of development is in-

188 ALBERT TPLER AND BRADLEY T. SCHEER

Control 15 30 45

cleavage fails to occur. In determining whether development could be completely and reversibly stopped by means of acid sea water it was necessary to determine first the lowest con- centration of acid in sea water in which cleavage failed. This for Dendraster eggs is a solution of about 2.5 cc. of 1.0 n HCl in 1000 cc. sea water. Eggs were transferred to such solu- tions at definite times after insemination and allowed to re- main in the solution for various times. The results with two sets are presented in table 5. It may be seen in set A that eggs transferred to the solution at 5 minutes after insemina- tion and returned to sea water 10 minutes later (15 minutes

45 Control 45 55 10 10 47 2 75 30 25 70 25

No cleavage 40 ' No cleavage

TABLE 6

Inhibition of development of Dendraster eggs in a d d sea water. Eggs transferred at 6 minates after insedmtion to: A, 8.5 cc. of 1.0 n HCI in 1000 cc.

sea water; B, 8.6 00. of 1.0 n HC1 in 1000 cc. sea water. Time in minutes after insemination.

A B I

Time of return Time of first 1 to seawater I cleavage

after insemination) show a delay of 10 minutes in the time of first cleavage. This would indicate complete cessation of development during the stay in the acid sea water. Develop- ment in all cases was found to be normal up to the prism stage. The eggs left 25 minutes in the acid gave, however, a 30-minute delay in cleavage and those left 40 minutes in the acid gave no cleavage at all. The results of set B are similar. It is evident, then, that for the stages investigated and the solutions employed development may be reversiblv blocked

ACID INHIBITION OF FERTILIZATION 189

PRODUCTION AND DEVELOPMENT OF TIGHT MEMBRANE EGGS

Moore ('30) showed that if unfertilized echinoid eggs are exposed to isosmotic non-electrolyte solutions (urea, glycerine or sugar) they develop after insemination without the forma- tion of fertilization membranes. Such eggs do not produce normal blastulae but give flattened plates of cells. The mem- brane forming capacity could be protected by the addition of certain electrolytes in the proper concentration. In a sub- sequent paper ('32) he noted that the addition of electrolyte (NaCI) in 'sub-optimal' concentration would allow the forma- tion of a membrane that was not raised from the surface. No information is given as to the development of these eggs.

Tight membrane eggs were found to owur spontaneously in varying amounts (1 to 2%) in some batches of untreated Dendraster and Lytechinus eggs. Tight membrane eggs were also produced by various treatments notably acid sea water.

A total of eighty-nine spontaneously occurring tight mem- brane eggs of Lytechinus and twenty of Dendraster were isolated and their development compared with normal eggs from the same lots. These eggs developed for the most part abnormally as the following figures show.

N o d plrctei A b n o d smbryos Tight membrane eggs 15 74

Controls 94 1 Lytechinus

Tight membrane eggs 8 12

Controls 22 0 Dendraster

Tight membrane eggs were produced in several ways. The following treatments gave both tight membrane and normal eggs so that control eggs could be isolated from the same treated lots. Shaking Dendraster eggs in a test tube immedi- ately after insemination for 1 minute gave about 80% tight membranes. From the isolation of forty-three such eggs and forty control eggs the following results were obtained.

Normal p2rcts.i Abnormal embryos Tight membranes 35 8 Control 30 10

190 ALBERT TYLER AND BRADLEY T. SCHEER

By treating unfertilized Lytechinus eggs with a solution con- taining 5 cc. of 1.0 n HCI per liter sea water for 2 minutes 5% of tight membranes were obtained and isolation gave:

Nonnal pltctsi Abnormal smbryos Tight membranes 10 28 Oontrol 50 0

Insemination in a solution containing 1.0 cc. of 1.0 n HC1 per liter sea water gave 10% tight membranes with Dendraster and 20% with Lytechinus eggs. Isolated eggs gave:

Normal plutai Abnormal smbryos Tight membranes 8 0

Controls 11 0 Dendraster

Tight membranes

Controls Lytechinus

39 12

92 16

One hundred per cent of tight membrane eggs is obtained in Dendraster by treatment at 15 seconds after insemination for '2 minutes with a solution containing 1.2 cc. of 1.0 n HC1 per 1000 cc. of sea water. In one such experiment practically all the eggs developed into normal plutei.

The early stages of development of the tight membrane eggs differ markedly from the normal. Figures 1 and 2 show normal unfertilized and fertilized eggs of Dendraster. In figure 3 a tight membrane egg is shown. In most cases the membrane is hardly distinguishable until cleavage occurs. At the 2-cell stage each blastomere instead of having the normal shape of a truncated spheroid (fig. 4) has the shape of a hemisphere (fig. 5). In the 4-cell stage each blastomere of the tight membrane egg (fig. 7) is an almost perfect quadrant of a sphere whereas in the normal egg (fig. 6) the cells are spheroids flattened on two sides. Cleavage continues in this fashion, the tight membrane restricting the separation of the blastomeres so that in the blastula stage the tight membrane egg has a much smaller blastocoel and much thicker wall than the normal egg. This condition is maintained after hatching and the blastula from the tight membrane egg (fig. 9) fails

ACID INHIBITION OF FERTILIZATION 191

to attain the ellipsoidal shape of the normal blastula (fig. 8). The difference in size remains until the end of gastrulation (figs. 10 and 11) then the tight membrane embryos gradually enlarge to the normal size. The tight membrane gastrula (fig. 11) appears to be filled with mesenchyme cells but there are probably only a normal number present. The smaller size of the blastocoel would cause the apparent crowding of the mesenchyme. When the pluteus stage is reached, em- bryos from the tight membrane eggs (fig. 12) are inas- tinguishable from the normal.

In the case of the spontaneously occurring tight membrane eggs development is generally the same up to the end of gastrulation then various kinds of abnormalities set in. The most frequent kind of abnormal embryo is one which does not proceed beyond the gastrula stage and at a time when the controls are in the pluteus stage it has formed a sort of exaggerated gastrula ; that is, the archenteron has pushed forward and a knob formed on the anterior end of the embryo. These ‘Dauergastrulae’ appear to be blocked in the gastrula stage and it would be of interest if a sufficient quantity could be obtained, to compare their metabolism with the normal embryos. Other abnormal embryos from spontaneously occurring tight membrane eggs may form skeletal rods and distorted plutei.

Just (’22) stated that when membrane elevation does not occur in typically normal fashion subsequent development is abnormal. The validity of this as a generalization for all forms that raise membranes was questioned by Tyler (’31) on the basis of the fact that normal embryos were obtained by artificial activation from Urechis eggs which did not ele- vate membranes until shortly before cleavage. However, when the membrane is finally raised it is indistinguishable from that formed after normal fertilization. In the case re- ported here the membrane remains closely surrounding the egg until hatching. It is evident that in spite of the failure of normal membrane elevation, and the consequent alteration in shape in the early stages, normal plutei are obtained. The

192 ALBERT TYLER AND BRADLEY T. SCHEER

failure to obtain normal plutei from spontaneously occurring tight membrane eggs must be due to other factors than the failure to elevate the membrane. Investigation was also made of the rate of development and rate of respiration of the tight membrane eggs. The results concern the question of the energetics of differentiation and will be reported in a subsequent publication.

DISCUSSION

That the period immediately following fertilization is one of high susceptibility to changes in the external medium has been shown in many experiments, notably those of Just ('22 to '29) with hypotonic sea water. That this difference in susceptibility is not the same with different agents is evident in the Urechis experiments as well as in the experiments of Harvey ( '30), Runnstrom ( '30) and Barron ( '32) in which fertilization could be obtained in anaerobic conditions but later development would be blocked in certain stages. There is no point in discussing the action of the acid sea water until more is known of the changes that occur upon fertilization and of the action of other agents. I t should, however, be noted that evidence has been obtained (Runnstrom, '30 et seq.) showing that an acid is produced upon fertilization in sea urchin eggs. It is conceivable that a certain amount of acidity of the medium may prevent the production of the acid in the egg. On the other hand, one might expect acid sea water alone to activate eggs parthenogenetically, and that is, in fact, the case as was first shown by Lefevre ('07) for strong acids. A n important step forward would be made if an agent were found that would in acid sea water overcome the effect of the acid.

From the facts, that the inhibiting concentration of acid sea water is roughly the same for the different forms investi- gated and that they all show a short susceptible period after insemination, one might conclude that the reactions under- lying the initial stages of fertilization are the same in all.

ACID INHIBITION OF FERTILIZATION 193

More information is needed, however, to justify such a con- clusion. It is entirely possible for different kinds of reactions to be blocked by the same change in pH.

As. was pointed out above it oould not be definitely con- cluded that a reversal of fertilization, as demonstrated in Urechis eggs occurred with the eggs of the various forms investigated here. Such an interpretation appears, however, to be the most likely one and it may be possible to demon- strate this by performing the experiments under different conditions, low temperatures for example.

SUMMARY

1. The concentration of acid sea water necessary to inhibit fertilization was determined for eggs of Paracentrotus, Sabellaria, Ascidiella, Leptonereis, Dentaliurn, Phallusia, Ciona, Dendraster, Lytechinus and Strongylocentrotus. The inhibiting concentrations vary around a solution of 1.2 cc. of 1.0 n HCl per 1000 cc. of sea water giving a pH of about 7.2. It cannot be concluded that significant differences existed in the different forms studied.

2. Fertilized eggs, placed in a stronger solution than neces- sary to inhibit fertilization, were found to develop normally. Development is blocked if the eggs are transferred to the acid sea water within 15 to 90 seconds after insemination.

3. Development proceeds normally if the blocked eggs are returned to normal sea water after a short exposure or if re-inseminated after a longer exposure. The most likely interpretation is that the entering spermatozoon is expelled and the establishment of the block to polyspermy is reversed by the acid sea water treatment.

4. Progressive retardation of development occurs in in- creasing concentrations of acid sea water but complete and reversible block could only be obtained for a short period of time.

194 ALBERT TYLER AND BRADLEY T. SCHEER

5. After certain treatments with acid sea water membrane elevation is prevented. The tight membrane eggs so pro- duced develop into normal plutei, although their early de- velopment is distinctly modified. Tight membrane eggs also occur in untreated batches of eggs, but in this case the de- velopment is generally abnormal, ‘Dauergastrulae ’ often resulting.

LITERATURE CITED

BAREON, E. S. c f . 1932 The effect of anaerobiosis on the eggs and sperm of sea-urchin, starfish and Nereis and fertilization under anaerobic condi- tions. Biol. Bull., vol. 62, pp. 46-53.

CLARK, J. M. 1936 An experimental study of polyspermy. Biol. Bull., vol. 70,

DRAOOIU, J., F. V d s AND Y. ROSE 1923 Consbquences cytologiques de l’abaisse- ment du pH extbrieur sur l’bvolution de l’oeuf d’oursin. C. R. Acad. Sci., T. 176, pp. 409-412.

The effect of lack of oxygez on the sperm and unfertilized eggs of Arbacia punctulata, and on ftLLilization. Biol. Bull., vol. 58,

JUST, E. E. 1922 Initiation of development in the egg of Arbaeia. I. The eff eet of hypertonic sea water in producing membrane separation, cleavage, and topswimming plutei. Biol. Bull., vol. 43, pp. 384401. 1929a Initiation of development in Arbacia. IV. Some cortical

reactions as criteria for optimum fertilization capacity and their significance for the physiology of development. Protoplasma, vol. 5,

1929 b Hydration and dehydration in the living cell. 11. Fertiliea- tion of the eggs of Arbacia in dilute sea water. Biol. Bull., vol. 57,

L ~ E V E E , GEORQE 1907 Artificial parthenogenesis in Thalassema mellita J.

MOORE, A. R. 1930s Fertilization and development without the fertilization membrane in the egg of Dendraster excentricus. Protoplasma, vol. 9,

Fertilization and development without the fertilization mem- brane in the egg of Strongylocentrotus purpuratus. Protoplasma,

The role of unantagonized cations in protecting the mem- brane forming function in the eggs of the sea-urchin. Protoplama, vol. 15, pp. 268-275. 1933 The relative values of cations in protecting the membrane

forming capaeity of the echinoids, Clypeaster japonicus and Temno- pleurus hardwiekii. Science Reports, Tohoku Imp. Univ., Series IV,

pp. 361-384.

HARVEY, E. B. 1930

pp. 288-292.

pp. 97-126.

pp. 443-448.

EXP. Zoiil., VOI. 4, pp. 91-149.

pp. 1-8. 1930 b

V O ~ . 9, pp. 9-17. 1932

V O ~ . 8, pp. 249-253.

ACID INHIBITION OF FERTILIZATION 195

RDNNSTRON, JOHN 1930 a Atmungsniechanismus und Entwicklungserregung bei dem Seeigelei.

1930 b Spaltung und Atmung bei der Entwicklungserregung des Seeigeleis. Arkiv. f . Zool., Bd. 21B, S. 1-5.

1924a The influence of hydrogcn-ion eon- centration on unfertilized Arbacia, Asterias, and Chaetoptereis eggs. Biol. Bull., vol. 47, pp. 304-322.

The influence of hydrogen-ion concentrations on the develop- ment of normally fertilized Arbacia and Asterias eggs. Biol. Bull., vol. 47, pp. 323-332.

The influence of hydrogen-ion concentration on the ferti- lization process in Arbacia, Asterias, and Chaetopterus eggs. Biol.

The production of normal embryos by artificial partheno- genesis i n the echiuroid, Urechis.

1936 On the energetics of differentiation. IV. Comparison of the rates of oxygen consumption and of development at diff ereut tempera- tures of eggs of some inariiie animals. Biol. Bull., vol. 71, pp. 82-100.

Inhibition and reversal of fertilization in the eggs of the echiuroid worm, Urechis caupo. J. Esp. Zoiil.,

Recherche5 sur le pH d’arret de la

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SNITH, H. W. AND H. A. CLOWES

1924 b

1924c

Bull., V O ~ . 47, pp. 333-344. TYLER, ALBERT 1931

Biol. Bull., vol. 60, pp. 187-211.

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division de l’oeuf d’oursin. C. R. Acad. Sci., T. 176, pp. 133-136.

T H E JOURNAL O F EXPERIMENTAL ZOOIAOOY, VOL. 75, NO. 2

PLATE 1

EYPLANATlON OF FIGUKES

I’hotoniirrographs of living eggs and embryos of Dendraster. Figures 1, 3, 4, Figures 3, .i, 7, 9, 11 and 12 are 6, 8 and 10 are of normal eggs and embryos.

of tight membrane eggs and embryos. 1 Unfertilized egg. 2 and 3 4 and 5 Two-cell stage. 6 and 7 Four-cell stage. 8 and 9

Fifteen minutes after fertilization.

Blastulae at about 1 hour after hatelling. 10 and 11 Completed gastrulae. 13 Pluteus.

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ACID INI I IRITIOPT O F F E R T I L I Z A T I O S ALBERT TYLER AND BRADLEY T. SCHEER

PLATE 1

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