Inhibition and reversal of fertilization in eggs of the echinoid worm, Urechis caupo

INHIBITION AND REVERSAL O F FERTILIZATION I N EGGS O F THE ECHINOID WORM.

URECHIS CAUPO

ALBERT TYLER AND JACK SCHULTZ Wm . G . Kerckhoff Laboratories of the Biological Sciences. California Institute of

Technology. Pasadena. California

ONE PLATE (FOUR FIGURES)

CONTENTS Introduction ........................................................ 509 The acid sea water experiments ...................................... 511

Inhibiting concentration of acid .................................. 511 The duration of the susceptible period ........................... 512 Changes upon fertilization in normal sea water mid upon transfer to

acid sea water .............................................. 513 The return to normal sea water ................................... 515 Fertilization of previously fertilized eggs .......................... 516 The third insemination ........................................... 519 Behavior of the sperm in the blocked eggs ........................ 520 Artificial activation of ‘fertilized’ eggs ........................... 520 Insemination in the acid solution .................................. 521

522 Sodium lactate and sodium citrate ................................. 522 Sodium iodo-acetate ............................................. 323 Other substances ................................................ 523

Discussion ............................................................ 524

Literature cited ...................................................... 528

Experiments with other agents ........................................

Summary ........................................................... 527

INTRODUCTION

I t is generally assumed that fertilization is an irreversible process-that once it has occurred. the egg cannot again be fertilized . Reversibility of the phenomenon is. however. re- garded as a possibility by at least one investigator in the field . Thus. Lillie (’19. p . 129) states: “The reactions of fertilization form an irreversible series though it is conceiv-

509

510 ALBERT TYLER A N D JACK SCHULTZ

able that some of them taken singly may be reversible.” In the work reported here evidence is presented that eggs of Urechis may be fertilized repeatedly and that at least a part of the fertilization reaction is reversible.

It has long been known that fertilization could be inhibited in marine eggs by acidifying the sea water. Smith and Clowes ( ’24) made careful determinations of the inhibiting concentration of acid in sea water for the eggs of several marine forms and found it to lie at pH 6.8 to 7.1. Loeb (’98) and others later had noted that eggs would divide in such solutions o r solutions even more acid than this, but that the velocity of division was generally lowered. Whether normal embryos would develop in solutions which inhibit fertilization was not ascertained.

The above work shows that fertilization is more readily inhibited than cleavage by changes in the pH of sea water. But it is well known that apparently normal cleavage does not always give normal development, and that processes oc- curring at fertilization influence later development. In order to single out the fertilization reaction for study, it is de- sirable to determine whether the agent used affects only that process and not the rest of development. This was done in the work reported here by noting the characteristics of the embryos which develop in the various solutions. It was found that perfectly normal embryos are obtained in solutions more acid than is required to inhibit fertilization, provided the eggs are transferred to such solutions at a definite time after fertilization.

The time after insemination in which fertilization could be inhibited by acid sea water was determined and found to be correlated with the occurrence of certain morphological changes associated with fertilization. It was observed in the early transfers of the eggs to acid sea water that these changes occurred and that the sperm entered. However, after a time, these eggs return in appearance to the unfertilized condition. When such eggs are later returned to normal sea water they do not resume development. I f , however,

FERTILIZATION I N EGGS O F URECHIS CAUPO 511

they are reinseminated in the normal sea water, fertilization occurs and development proceeds. The cleavage of such eggs is generally polyspermic, showing that the spermatozoa from both the first and second insemination take part in development.

The eggs used in these experiments were those of the marine worm, Urechis caupo (Fisher and MacGinitie, '28).

Evidence was also obtained that solutions of sodium citrate and sodium lactate in sea water, at the pH of normal sea water, give results essentially similar to acid sea water in regard to inhibition and reversal of fertilization. A variety of other substances, such as formaldehyde, KCN, phenylurethane, nicotine, sodium acetate, sodium iodo-acetate, and sodium chloride in sea water, were found to inhibit fertilization only in concentrations in which fertilized eggs developed very abnormally or not at all.

T H E ACID SEA WATER EXPERIMENTS

Inhibiting concerztration of acid The concentration of acid necessary to inhibit fertilization

in eggs of Urechis was found to be very low. For the determination of the inhibiting concentrations a stock solution of 0.001 molar HC1 in sea water was prepared, shaken to drive off excess CO, and allowed to stand in contact with air. From this a series of concentrations was made up by dilution with sea water. A drop of eggs containing less than 0.03 cc. of sea water was added to 10 cc. of each of the solutions employed. The eggs were allowed to remain in the solution for at least five minutes and then inseminated with a drop of sperm.

I n table 1 the results of one such experiment are presented. I n the solutions more concentrated than 0.0003 molar HC1 in sea water fertilization does not occur. The concentration giving complete inhibition may be taken to lie between 0.0003 and 0.0004 molar. The hydrogen-ion concentration of these solutions determined colorimetrically was approximately pH 7.1 to 7.2 and the pH of normal sea water was 8.2. Four sets

512 ALBERT TYLER AND JACK SCHULTZ

of experiments on different batches of eggs gave inhibiting concentrations ranging from 0.00035 to 0.0005 molar HCI.

In another series of experiments iodo-acetic acid was used, and the inhibiting concentration was found to lie within the same range as for the HCI.

The duration, of the susceptible period After the determination of the strength of solution neces-

sary to inhibit fertilization, transfers were made to the acid sea water at various intervals after insemination. The acid sea water solution used was sufficiently acid to block fertilization completely. It was found that eggs transferred soon after insemination did not develop, whereas later transfers

TABLE 1

Inhibiting concentration of HC1

HCl IN SEA WATER FERTILIZATION, CLEAVAQE, MOLARITY PER CENT PEE O l N T

0.0000 0.0001 0.0002 0.0003 0.0094 to 0.0010

showed the same amount of normal development as the controls.

Table 2 gives the data for a typical series of experiments. The eggs were washed and inseminated in normal sea water at one-half hour after removal from the animal. Samples were then transferred with less than 0.03 cc. of the sea water to dishes of acid sea water at the time intervals stated in the table. The table gives the percentage of fertilization and the percentage of the fertilized eggs that give normal and abnormal two-day embryos. The amount of fertilization is given as the percentage of eggs in which germinal vesicle breaks down. As will be pointed out later, this does not mean that where 0 per cent fertilization is listed the sperm did not enter.

FERTILIZATION IN EGGS O F U R E C H I S CAUPO 513

It is clear from the table that eggs transferred earlier than two and one-half minutes after insemination show no fertilization. For the eggs transferred later, successively higher percentages of fertilization are obtained. The percentage of normal development likewise increases, until at ten minutes it is practically as high as in the controls. Of these eggs which develop, in the later transfers, a certain percentage (37 per cent of the total eggs, in the three-minute transfer of table 2) behave like the poorly activated eggs previously described (Tyler, '31 a). They do not elevate membranes, and

TABLE 2

Duration of susceptible period. Experiment of 4/36/58. Temperatwe, 19.5"C. Solution = 0.001 molar HCl in sea water

TIME AFTER INSEMINATION, MINUTES

Control (normal sea water) 1 minute 30 seconds 2 minutes 2 minutes 30 seconds 3 minutes 4 minutes 5 minutes 7 minutes

10 minutes

FERTILIZATION, PER CENT

100 0 0 18 7 7 94 98.5 100 100

NORMAL EMBRYOS

ABNORMAL EMBRYOS

95 0 0 1

30 85 60 40 90

5 0 0

10 35 10 25 30 10

remain indented for some time, although the germinal vesicle disappears.

Six other full experiments of this type and many other incidental repetitions gave similar results. Normal embryos are obtained in solutions which are too acid for the occurrence of fertilization. This was found to be true for solutions as acid as pH 6.4, if eggs are transferred to such solutions later than three minutes after insemination.

Three minutes after insemination appears to represent a critical stage in the development of the egg. Eggs transferred to the acid sea water prior to this time are blocked, whereas later transfers to the same solution give normal embryos.

514 ALBERT TYLEH. AND JACK SCHULTZ

Changes upon fertilization in normal sea water and upon t rans fer t o acid sen water

The changes that occur in these first three minutes after normal insemination are thus of especial interest. These changes have been described in detail in previous publica- tions (Tyler, '31 a, '32 b). The egg of Urechis is in the germinal-vesicle stage before fertilization and possesses a Iarge indentation which marks the pole of the egg (Tyler, '32 a ) . Within two and one-half to three minutes after insemination the indentation disappears, the egg becomes spherical, and the germinal vesicle is drawn out toward the pole. During this time the sperm is attached to the egg which raises a small protuberance below the membrane. This enlarges and forms a striking clear process (the fertilization cone) which engulfs the sperm. There is some variability in the time at which the sperm passes from the fertilization cone into the egg, but this generally occurs just at or very shortly after the time of disappearance of the indentation. After this time the germinal vesicle breaks down, the fertilization membrane is elevated, polar bodies are extruded, etc.

Observations were made on living eggs t ransfcrred within two minutes to the acid sea water in order to determine which of the early changes took place. It was seen that the rounding out of the egg and drawing out of the germinal vesicle toward the pole continues in the acid sea water. This is ilIustrated in figures 1 and 2. I t was also seen that the fertilization cone formed and engulfed the sperm which then entered the egg. Subsequent to this the egg is then seen to become indented again and to assume the appearance of the unfertilized egg (figs. 3 and 4). Cytological preparations of such eggs showed that the sperm remains within the egg.

The time schedule of events is slowed up in the acid sea water, When the eggs are transferred to the acid sea water a t one minute after insemination the rounding out of the egg and entrance of the sperm is delayed until ten to twelve minutes, instead of three minutes after insemination. The return of the egg to its original appearance then requires

FERTILIZATION I N EGGS O F U R E C H I S CAUPO 515

Control (in normal sea water) 2 minutes 10 seconds 4 minutes 6 minutes 8 minutes

10 minutes 12 minutes 14,16,18,21, and 26 minutes

about ten minutes for completion. Thus, when the eggs are transferred to the acid sea water at one minute after insemination they become 'fertilized' and then return to a condition similar in appearance to that of the unfertilized egg.

100 85 15 96 5 70 89 80 10 78 5 25 82 0 12 93 2 14 26 15 10

0 0 0

The return t o %normal sea water

The question now arises whether upon return to normal sea water development is resumed. This was investigated in a number of experiments. The results showed that for eggs transferred to acid sea water within three minutes after insemination, development is resumed provided the eggs are

TABLE 3

The return to normal sea water. Experiment of 4/66/58. Temperature, 19.5"C.

NORMAL ADNORMAL EMBRYOS,

PER CENT PER CENT

TIME AFTER INSEMINATION, FERTILIZATION. EMBRYOFJ, MINUTES PER CENT

returned to normal sea water in less than ten minutes. How- ever, if the eggs are returned to normal sea water at various times later than ten minutes after insemination, successively fewer develop until at twenty minutes after insemination none of the eggs resume development.

I n table 3 are presented the results of one series of experiments in which eggs are returned to normal sea water. The eggs were first transferred to the acid sea water at one minute after insemination. Samples were then returned to normal sea water at the time intervals stated in the table. The usual precautions in regard to the amount of solution transferred with the eggs, etc., were taken. It may be noted

THE JOURNAL O F FHPERIMENTAI. Z O O L O G Y , VOL. 63, N O , 2


that for the shortest treatments with acid sea water the percentage of eggs which resume development approximates that of the controls. However, for the eggs returned to normal sea water at ten or more minutes after insemination the percentage of fertilization falls off progressively. Thus the eggs that were left in the acid sea water for thirteen minutes (fourteen minutes after insemination) showed no resumption of development.

The percentage of normal embryos obtained in the experiment detailed in table 3 approximates that of the controls in only one instance, namely, the transfer made at four minutes after insemination. However, in other less extensive experiments the proportion of normal embryos obtained in transfers up to five minutes after insemination was practically the same as for the controls. It was also noted that the cleavage of the eggs which resumed development upon return to normal sea water was generally normal; that is, no ap- preciable polyspermy was observed.

A comparison of the results presented here and the time schedule of events given above shows that the failure to resume development begins at the time of rounding out of the egg in the acid sea water and that the time of treatment necessary for complete inhibition coincides with the time in which the egg is returning to its unfertilized appearance.

Fertilization of previously fertilized eggs The observations on the living eggs showed that the sperm

enter the eggs in the acid sea water. It was therefore deemed important to investigate the possibility of refertilizing such eggs. Since the eggs which are treated with acid sea water for thirteen minutes or more show no development upon return to normal sea water, an exposure of a t least that length of time was used for the experiments reported here. The results of many experiments on reinsemination showed that all of the blocked eggs are capable of fertilization. The eggs respond perfectly normally to the second sperm, but the first division generally gives three or four cells which is typical

FERTILIZATION I N EGGS O F URECHIS CAUPO 517

L E N U T H O F ACID

TREATMENT, MINUTES

Control 13 15 17 20 25

of dispermic eggs. Thus, both the first and second spermata- zoa participate in the development of these eggs. The development is, of course, generally abnormal.

In table 4 the results of experiments of this type are presented. The eggs were first inseminated in normal sea water and then transferred one minute later to the acid sea water. They were allowed to remain in the acid sea water f o r the time intervals stated in the table and then samples were returned to normal sea water. These were then inseminated with a dilute suspension of sperm. At the same time a sample of untreated eggs was inseminated with the same amount of

TABLE 4

Reinsemination. Experiment of 4/26/32. Temperature, 19.5"C.

TYPE O F FIRST CLEAVAQE TYPE OF EMBRYOS

2EE& 3 Cells, 4 Cells, Normal, Abnormal, Dead, per cent per ceT;t per cent per cent per cent per cent per cent

80 0 10 10 85 15 0 5 6 78 11 10 85 5 6 4 84 6 5 85 10 9 6 83 5 2 78 20

13 10 74 3 1 84 15 7 2 90 1 1 89 10

sperm suspension. Another control, not listed in the table, was run in many of the experiments: unfertilized eggs were treated with acid sea water for twenty minutes or more, then returned to normal sea water and inseminated with the usual amount of sperm. Eggs so treated show typically normal cleavage and development.

Practically all of the blocked eggs are capable of refertilization. Their cleavage is that of dispermic eggs. The eggs treated with acid sea water for thirteen minutes or more at one minute after insernination give 100 per cent fertilization upon insemination in normal sea water, and an average of 87 per cent of them divide into three or four cells. The controls in the case listed in the table showed 20 per cent


polyspermy. This is exceptionally high, but the data of this experiment are presented, inasmuch as they were obtained from the same batch of eggs as was used in tables 2 and 3. These eggs were removed in the morning and used for the experiment of table 4 in the afternoon. As is well known, the susceptibility of eggs to polyspermy increases with their age. It is therefore not surprising that as high as 20 per cent polyspermy was obtained in the controls. This, however, does not interfere seriously with the interpretation of the results, inasmuch as the percentage of dispermic cleavage obtained in the reinseminated eggs is more than four times as large as that in the controls. Further, in many other experiment s the controls showed practically no polyspermy, whereas that in the reinseminated eggs was very high. I n the average of six experiments the controls gave 7 per cent polyspermic cleavage, whereas the acid-treated eggs gave 77 per cent of polyspermic cleavage upon reinsemination.

Variation in the length of the acid treatment apparently has no effect on the extent of polyspermy that occurs after reinsemination. Such differences as are noted in table 4 in the percentage of four-cell cleavage and of normal embryos are very probably not significant, inasmuch as they are not found in other experiments. I n some experiments the time after insemination before transfer to acid was varied from one-half to two and one-half minutes, but the results upon subsequent fertilization in normal sea water were substanti- ally the same in all cases.

I n this connection, it should be noted that Smith and Clowes ( '24), and Rose ( '30) obtained a high percentage of polyspermy by inseminating sea-urchin and starfish eggs in solutions slightly less acid than those required to block fertilization. The possibility that the acid sea water treatment alone might be responsible for the polyspermy observed in the experiments reported here was therefore carefully examined. The fact that eggs which are inseminated in normal sea water, transferred to acid within three minutes, and returned within ten minutes to normal sea water showed practically no poly-

FERTILIZATION I N EGGS OF URECHIS CAUPO 519

spermic cleavage, is in itself evidence against such an assumption.

It should further be noted that eggs of Urechis which are inseminated in normal sea water, transferred to acid sea water within three minutes, and returned to normal sea water thirteen minutes later, show a number of extra sperm attached to their surfaces. It may be asked why these extra sperm do not fertilize the eggs on the return to normal sea water; in other words, why a second insemination is necessary. The result means that the attached sperm are rendered incapable of fertilization either by the initial action of the egg in blocking polyspermy or by the action of the acid sea water on the sperm itself or on its reaction with the egg. It is not known whether the extra sperm attached to normally fertilized eggs are functional for fertilization. This could be determined by the removal of such sperm from the eggs to which they are attached, and their addition to unfertilized eggs.

A number of experiments make it certain that prolonged treatment with acid sea water does not irreversibly affect eggs and sperm separately; that is, they are both capable of fertilization and normal development in sea water. In one experiment sperm and eggs were treated separately for fifteen minutes with acid sea water, and inseminated in the acid solution. The eggs were transferred three minutes later to normal sea water and gave 80 per cent normal cleavage. The sperm were attached to the eggs in the acid sea water; however, it is possible that those which initiated development were still free swimming in the solution.

The third iwsemiwation

The eggs which are fertilized in normal sea water after previous inhibition by acid sea water can be blocked a second time by the same technique. If they are transferred to acid sea water within three minutes after the second insemination the same behavior is observed as occurs in the first inhibition by the acid solution. The second sperm may be seen to enter


and the above-described changes in shape take place. When later returned to normal sea water and inseminated, the eggs give a normal fertilization reaction. The first cleavage then gives five, six, seven, or eight cells as is to be expected from trispermic eggs.

Behavior of the sperm iw the blocked eggs

Cytological preparations of acid-treated eggs were made by the method previously described (Tyler, '32 c). The eggs were transferred to acid sea water at one and one-half minutes after insemination, and samples removed for preservation at various time intervals. An examination of the slides showed that the spermatozoon which enters does not penetrate far into the egg even after thirty minutes in the acid sea water, but remains unchanged near the periphery.

Preparations were also made of the eggs which had been returned to normal sea water and reinseminated. In many such eggs two sperm were clearly visible, and the formation of the typical dispermic cleavage figure could be followed through the successive stages.

The eggs which were inhibited a second time by means of the acid sea water and again inseminated in normal sea water showed three sperm, which undergo their transformation into pronuclei and chromosomes at practically the same rate. This was also observed in the case of the dispermic eggs, and is to be expected from the fact that the sperm undergoes no apparent change in the egg while in the acid sea water.

Artificial actiuatiow of 'f ertilixed' eggs

Since it was found that the sperm actually enter the eggs whose development is blocked by acid sea water, the possibility of artificial activation of these eggs was investigated. The inhibited eggs were treated with hypotoiiic sea water, which has been shown to be an effective agent for parthenogenesis in Urechis (Tyler, '31 a, b). It was indeed found that such eggs respond to dilute sea water treatment. But


their development is that of fertilized rather than parthenogenetic eggs.

I n one experiment the eggs were transferred to acid sea water at one and one-half minutes after insemination. They were returned to normal sea water fifteen minutes later, and as usual presented the appearance of unfertilized eggs. A sample was left for control, another sample reinseminated and a third sample treated with dilute sea water. The reinseminated eggs gave 80 per cent polyspermic cleavage, showing that the first sperm had entered most of the eggs. The eggs treated with dilute sea water gave 70 per cent activation, 50 per cent cleavage, and 15 per cent normal embryos. This percentage of normal embryos is much greater than is obtained in the straight parthenogenesis experiments (Tyler, '31 a). The results make it probable that the sperm present in the inhibited eggs take part in their development after artificial activation.

Inseminatiorz in the acid soJution The question arises whether sperm can enter eggs in acid

sea water of the inhibiting strength. It was found that when unfertilized eggs are allowed to remain in acid sea water for some time, inseminated, and later removed to normal sea water, a second insemination in the normal sea water gave normal monospermic cleavage and normal embryos. For example, in one such experiment, the eggs were inseminated in 0.001 molar HC1 in sea water after a sojourn of one hour in that solution. They were removed to normal sea water forty minutes later and reinseminated at that time. All of the eggs were fertilized and at the first cleavage all divided into two cells, as did the controls which had been inseminated at the Fame time. Ninety-five per cent of normal top-swimming embryos were obtained from the treated eggs as compared with 90 per cent in the controls. A number of experiments of this type gave similar results. In some instances, however, sperm were seen to enter eggs which were inseminated in the acid sea water. Whether this occurs only when the eggs


are inseminated immediately after immersion in the acid was not definitely determined.

It is clear from these experiments that sperm do not enter eggs which are allowed to remain in acid sea water for some time before insemination. It is also evident that the acid sea water treatment used here does not render the eggs susceptible to polyspermy.

EXPERIMENTS WITH OTHER AGENTS

Sodium lactate and sodium citrate Of a number of other substances employed, sodium lactate

and sodium citrate were found to give results similar to those obtained with the acid sea water, although the pH of the solutions used was approximately the same as that of normal sea water.

The concentration of sodium lactate in which Urechis eggs do not fertilize is approximately 0.01 molar in sea water. This solution was made up isotonic with normal sea water and colorimetric determination of the pH gave a value of 8.2. When eggs are transferred to such a solution within two minutes after insemination, fertilization is blocked, whereas transfers made later than that time show normal cleavage and development just as in the case of the acid sea water. The blocked eggs were later returned to normal sea water. They were all found to be capable of fertilization and gave a high percentage of polyspermic cleavage, showing that the sperm from the first insemination had entered. Eggs inseminated in the lactate solution, transferred to normal sea water and roinseminated, gave typically normal cleavage and development indicating that the first sperm had not entered, again paralleling the results of the acid sea water experiments.

The sodium citrate blocks fertilization at a concentration of 0.02 molar in sea water. The inhibition and refertilization experiments gave results quite comparable with those of the lactate and the acid sea water experiments.


Phenylurethane Formaldehyde Potassium cyanide Sodium acetate Sodium chloride

Sodium iod o-acetate

Some of the first experiments on the inhibition of fertilization were carried out with iodo-acetic acid. As was noted above, this acid was effective in the same concentrations as the HC1. Experiments were also performed with the salt of this acid. It was found that, whereas a 0.0003 molar solution of iodo-acetic acid in sea water inhibits fertilization, a 0.02 molar solution of sodium iodo-acetate produces no such effect. However, in spite of the fact that 100 per cent fertilization and cleavage occurs, the eggs develop abnormally, even in concentrations as low as 0.0001 molar.

0.00005 0.002 0.01 0.02 (4 parts 0.5 molar NaC,H,O, to 6 parts sea water) 0.02 (4 parts 0.5 molar NaCl to 6 parts sea water)

TABLE 5

Concentrution reqwired to inhibit fertilization

SUBSTANOE MOLARITY IN SEA WATEE I

It has been demonstrated that lactic acid is produced in the metabolism of Arbacia eggs (Perlzweig and Barron, '28). A body of evidence (Lundsgaard, '30; Dudley, '31, etc.) shows that the breakdown of glucose to lactic acid is prevented in muscle and yeast by iodo-acetate. If the lactic acid is produced in eggs by a similar mechanism to that in muscle and yeast, then the present results indicate that such a system is not concerned with the fertilization process. Whether lactic- acid production in eggs is prevented by iodo-acetate remains to be determined.

Other substalzces A number of experiments were carried out with the sub-

stances listed in table 5, to determine their effects on fertilization. The concentrations required to inhibit fertilizat,ion are given in the table. In every case when eggs are trans-


ferred to such solutions at three or more minutes after fertilization, development is either arrested or continues abnormally. Even in weaker concentrations in which fertilization is obtained, subsequent development is abnormal. This is just the reverse of the results obtained with acid sea water, sodium lactate, and sodium citrate. The inhibition by sodium acetate and sodium chloride appears to occur at the same concentration. This may be due to similar disturbances in the ionic balance of the solutions.

Lillie ('21) was able to inhibit fertilization in Arbacia by transferring eggs to a weak solution of copper chloride in sea water within a few seconds after insemination. But here again no normal embryos are obtained in transfers made at a later time after insemination.

DISCUSSION

The results presented here may be taken to mean that the initial portion of the fertilization process is reversible. Thus, eggs which have received one spermatozoon will receive a second one if the reaction attendant upon the entrance of the first sperm is blocked in time. The solutions employed not only block fertilization, but evidently cause the egg to return to its unfertilized condition.

It might be claimed that the 'real' fertilization reaction does not take place in the acid sea water o r in the citrate or lactate solutions. Thus Lillie ( '19, p, 131) states :

It is usual to regard penetration of the spermatozoon as synony- mous with fertilization, but we may have fertilization well begun after mere attachment of the spermatozoon and without any penetration, as in the case of Nereis; on the other hand we may have penetration without any fertilization if the egg is not in the proper condition. A complete fertilization reaction involves penetration which thus furnishes one of the problems of fertilization; but, is not fertilization in itself.

Penetration without the occurrence of the fertilization reaction has been noted very early by 0. and R. Herlwig ('87) for unripe sea-urchin eggs treated with chloral hydrate and by Lillie ('19) for unripe eggs of Cliaetoptems.


But such objections cannot be raised to the interpretation presented here. It is evident that the fertilization reaction in Urechis starts with the attachment of the sperm. It is, therefore, that part of the reaction of the egg which occurs within the first three minutes after insemination that is capable of being reversed by means of acid sea water, lactate or citrate solutions.

I n artificial parthenogenesis, a few cases interpreted as demonstrating reversibility have been reported. Loeb ( '13, '15) claimed to have been able to fertilize the blastomeres of artificially activated Strongylocentrotus eggs. But Moore ('17) was unable to confirm this result on isolated blastomeres of artificially activated Arbacia eggs. Loeb also reported that eggs in which membrane elevation had been artificially induced could be fertilized upon removal of the membrane. This point was carefully investigated by Moore ('16, 'li), Lillie ( %), and Just ( '19, '22), as was also the question of fertilization of eggs in which mitotic changes had been artificially induced. They concluded from their results that fertilization does not occur in eggs which have been given an optimum exposure to the artificial agent. Such experiments do not bear on the question of reversibility, since at most they demonstrate a retention of the ability to respond to sperm on the part of eggs which have been insufficiently activated.

Bury ('12) described an experiment in which eggs were placed at 0°C. thirty to forty minutes after fertilization. On reinsemination, at the low temperature, new sperm were stated to have entered. The most probable interpretation of these results is that the physical condition of the egg is altered by the low temperature; they do not therefore necessarily bear on the problem of reversibility.

Another experiment reported by Loeb ('13) is more closely related to the problem. He found that eggs of Arbacia, treated with various activating agents, might be induced to 'return' to their resting, unfertilized condition by exposure to sodium cyanide or chloral hydrate. When sperm were added to such eggs after their return to normal sea water, development followed and normal blastulae were produced.


The objection has been raised (Lillie, ’19, p. 165) that no visible changes occur in the solution of the activating agent, so that the only reversal involved concerns that change in the egg which causes its activation upon return to normal sea water. A ‘reversal’ of this sort has been noted by Tyler (’31b) in eggs of Urechis which have simply been overex- posed to dilute sea water. They resume their unfertilized appearance upon the return to normal sea water, can be fertilized and produce normal embryos, although a shorter treatment would have resulted in 100 per cent artificial activation.

In the experiments reported here, the occurrence of a ‘reversal’ is unquestionable . Whether it is a part of the fertilization reaction that is reversed is a matter of definition. That the block to polyspermy is established almost instaneously is a truism of embryology. The beginning of the fertilization reaction in normally monospermic eggs may be defined properly as the establishment of the block to polyspermy. If this be granted, it is apparent that the changes which can be reversed in Urechis eggs are part of the fertilization reaction. On this basis it may be claimed that a part of the fertilization reaction can be reversed.

The limit beyond which reversal does not occur by treatment with acid sea water was found in the, present experiments to coincide with the completion of the rounding up of the egg and the beginning of membrane elevation. The possibility of reversal of later stages remains open.

It may be objected that a complete reversal should involve an expulsion of the sperm from the egg. However, it is claimed only that the reactions of the egg, such as the morphological changes attendant on fertilization and the establishment of the block to polyspermy, are reversed. The return of the egg to a condition in which it can be fertilized occurs in spite of the presence of a spermatozoon within it. The egg in this condition does not permit the normal transformations of the entered sperm into the male pronucleus, even after the return to normal sea water. After fertilization by a second sperm, however, both spermatozoa undergo the usual changes at the same rate.

FERTILIZATION IN EGGS OF U R E C H I S CAUPO 527

The manner in which the acid sea water produces its effect may now be briefly considered. It is known that lowering the pH of sea water retards cleavage (Loeb, '98, and others). This was found to be true for Urechis eggs transferred to the acid solution later than three minutes after insemination. It is also known that the oxygen consumption of sea-urchin eggs drops in acid sea water (Warburg, '10). But it is doubtful whether the acid sea water produces its effect through the lowering of the oxygen consumption, since KCN, which is also known to lower oxygen consumption (Warburg, 'lo), does not produce the same results. The results of Barron ('32), who found that fertilization occurred in Nereis after both eggs and sperm had been kept in anaerobiosis for five hours, may also be mentioned.

It is possible that the acid sea water may act through its effect on the viscosity or permeability of the egg. But although it is known that acids influence these properties of living cells (see, for example, Heilbrunn, '28), in the absence of specific data on Urechis eggs there is no advantage at present in a discussion of this aspect of the problem.

Whatever the effect of the acid, the comparable results obtained with sodium citrate and lactate indicate a similar action on the egg. Since they are salts of weak acids, their action may be due to the penetration of the undissociated acid and the consequent change in the intracellular pH, although it should be noted that sodium acetate behaves differently.

SUMMARY

1. Fertilization can be blocked in eggs of Urechis caupo by means of acid sea water of pH 7.1 to 7.2.

2. The block may be established up to three minutes after insemination, so that eggs transferred to the acid solution before that time show no development, whereas eggs transferred later than that time give perfectly normal embryos.

3. Development is resumed in the acid-blocked eggs if they are returned to normal sea water within ten minutes after the transfer to the acid solution. If returned at a later time they do not resume development.


4. Eggs placed in acid sea water at one minute after insemination continue with the normal changes in shape that occur upon fertilization and the sperm enter. But these processes require about ten minutes rather than three for their occurrence. The eggs then return to their unfertilized appearance about ten minutes later.

5. The acid-blocked eggs which do not resume development upon return to normal sea water can all be fertilized with fresh sperm. They give a normal fertilization reaction, but, due to the presence of two sperm in each egg, they divide into three or four cells at the first cleavage and produce mainly abnormal embryos.

6. The eggs can be fertilized a third time if they are transferred to acid sea water within three minutes after the second insemination and returned to normal sea water more than ten minutes later. They then behave as trispermic eggs.

7. A cytological examination of the repeatedly fertilized eggs shows that the two (or the three in the case of three in- seminations) spermatozoa undergo their transformations into pronuclei at approximately the same rate.

8. Eggs in which fertilization has been blocked can be activated artificially and behave as fertilized rather than parthenogenetic eggs.

9. Sodium lactate and sodium citrate at concentrations of 0.01 and 0.02 molar in sea water, respectively, give results similar to those with acid sea water. Sodium acetate, sodium chloride, sodium iodo-acetate, phenylurethane, formaldehyde, and potassium cyanide act differently from the acid sea water.

10. The results of this work are taken to demonstrate that the initial part of the fertilization reaction is reversible.

LITERATURE CITED

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LOEB, JACQUES 1898 Ueber den Einfluss von Alkalien und Sauren auf die embryonale Entwicklung und dns Wachstum. Arch. Entwmech., Bd. 7, 8. 631.

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PLATE 1

EXPLANATION OF FIGURES

Photomicrographs of the same living egg, in the same position, showing changes Insemination performed in

Indentation still present. Sperm (q.) may be seen on the periphery, about

in shape and entrance of sperm in acid sea water. normal sea water, eggs transferred after one minute to the acid sea water.

t o enter the egg. 1

2 Egg rounded out. Sperm almost completely entered. 3 and 4 Successive stages in the return of the indentation.

FERTILIZATION I N EGGS OF URECHTS CAUPO .iI .TIERT TY1,IClG .4Xn J A C K SCHUI.TZ

Inhibition and reversal of fertilization in eggs of the echinoid worm, Urechis caupo

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