The respiration and fertilizable life of Arbacia eggs under sterile and non-sterile conditions

THE RESPIRATION AND FERTILIZABLE LIFE O F ARBACIA EGGS UNDER STERILE AND

NON-STERILE CONDITIONS

ALBERT TYLER, NELDA RICCI AND N. H. HOROWITZ William G. Kerclchoff Laboratories of Biological Sciences,

California Institute of Technology, Pasadena '

T W O FIGURES

Considering the unfertilized egg as a 'resting cell,' exhibiting simply maintenance metabolism, one might expect its respiratory rate to be low in comparison with a developing egg and to be relatively constant with time. That the rate is not always lower before than after fertilization is clear from the work of Whitaker ( '33). Nor does the rate appear to remain constant but rises with time as Warburg ('14) and Runnstrom ('28) have shown. This rise appeared to be correlated with loss of fertilizability according to Tyler and Humason ('37) since in species where the fertilizable life is short the rise in respiratory rate is found to set in sooner.

The starting point for the experiments reported here was an attempt to test this correlation by measuring the respiration in the presence of agents such as alcohol (Whitaker, '37) that are known to prolong the fertilizable life. Such experiments have been performed by Runnstrom ('28). He found that the spontaneous rise in respiration of unfertilized sea urchin eggs is inhibited by carbon monoxide. The carbon monoxide is also shown to extend the fertilizable life of the eggs. Our experiments with alcohol confirm the results of 'This investigation was supported in part by a grant from the Penrose fund

The experiments were performed at the We are indebted to

of the American Philosophical Society. Marine Biological Laboratory, Woods Hole, Massachusetts. Dr. S. A. Waksman for his advice concerning methods for marine bacteria.

129

130 A. TYLER, N. RICCI AND N. H. HOROWITZ

Runnstrom with carbon monoxide, but by following bacterial growth during the experiments it appeared probable that it is with this factor that the rise in respiratory rate is correlated.

The question was further investigated by measuring the respiration of unfertilized eggs under sterile conditions and by measuring bacterial respiration alone. It became of interest then to determine whether the presence of bacteria influ- enced the length of life of the unfertilized egg. Evidence for this was presented some time ago by Gorham and Tower ('02) whose conclusions were subsequently questioned by Loeb ( '11). We therefore examined the effect of sterile conditions, of excess bacteria and of alcohol on the life span of the unfertilized egg.

MATERIAL AND METHODS

Eggs of Arbacia punctulata were used in these experiments. The oxygen consumption was measured in Warburg manom- eters using the cylindrical type of vessel previously described (Tyler, '36), but of 18 to 20 cc. calibration volume. In dis- tributing the eggs to the vessels, precautions were taken to insure uniform sampling. In most cases egg counts were made of a diluted sample of the suspension.

The bacterial counts were made from serial dilutions of the egg suspensions plated out in sea water agar medium (Waks- man et al., '33). The egg suspensions were vigorously shaken before plating out. This served to fragment the eggs and distribute bacteria that might adhere to the surface, as well as to make sampling from suspensions of intact eggs comparable with that from disintegrated eggs.

EFFECT OF ALCOHOL

Whitaker ('37) showed that 1% ethyl alcohol in sea water would prolong the fertilizable life of Urechis eggs to about three times the control span. We could confirm this effect with Arbacia eggs ; a 2% solution giving the best results which amounted to approximately double the control fertilizable life.

RESPIRATION AND LIFE SPAN O F EQGS 131

Measurements of the oxygen consumption in 2% alcohol showed that while the initial rate is the same as that of the controls, the subsequent rise in rate is greatly retarded. I n a typical experiment with three control and three alcohol vessels the following figures were obtained for the oxygen consumption (cubic millimeters per hour).

In 8e.i~ water In 2 % alcohol 2nd hour 15.7 13.6 13.3 12.2 11.5 14.9

10th hour 22.8 23.5 21.7 15.5 16.8 16.6 22nd hour . . . 168.0 219.4 . . . 44.8 56.2

Samples of the eggs from one pair of vessels, inseminated after the tenth hour, gave 90% cleavage for the alcohol treated lot and 60% for the controls. At the twenty-second hour the control eggs were about 95% cytolyzed or disintegrated, and about 1% of the apparently intact ones divided after insemination. The eggs from the alcohol vessel at this time showed about 10% cytolysis or disintegration, and 40% of the intact eggs divided.

The results of another experiment are presented graphically in figure 1. As is evident from the figure the rise in respiration is considerably delayed in the 2% alcohol. In this experiment one pair of vessels was removed at 20 hours, another at 25 hours and the third pair at 38 hours for examination and insemination of the eggs, and for plating out for bacterial counts. The figures for the number of bacteria are given at the corresponding points on the graphs. Examination of the eggs from the vessels after insemination gave the following percentages of cleaved (C), intact uncleaved (I) and cytolyzed or disintegrated eggs (D).

In 8ea water I n 8 % alcohol C I n C I T)

20 hours 15 45 40 90 5 5 25 hours 5 20 75 85 5 10 38 hours 0 15 85 0 60 40

It is clear here that the rise in respiration is accompanied by cytolysis and disintegration of the eggs. However, the bacteria increase considerably in numbers at the same time, and

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132 A. TYLER,, N. RICCI AND N. H. HOROWITZ

in the alcohol the increase is considerably slower than in the sea water as the figures show. The figures do not show a di- rect relation between the increase in number of bacteria and the rise in respiratory rate. For example, in the sea water vessels there is an eighty-sixfold increase in bacteria between the twentieth and thirty-eighth hour while the rate of oxygen consumption has gone up only 74 times (taking 40 cu.mm. and 305 cu.mm. as the excess respiration at 20 hours and 38 hours

Fig. 1 Rate of oxygen consumption of unfertilized Arbacia eggs in sea water containing 2% alcohol and in ordinary sea water. The figures on the graph give the number of bacteria per cubic centimeter in the vessels removed at 20, 25 and 38 hours.

respectively). This might be taken to mean that the excess respiration is not due entirely to bacteria. However, we can- not expect the bacteria to respire a t a constant rate in an experiment of this sort in which the cultural conditions are changing radically due to progressive cytolysis and disintegration of the eggs as well as to the accumulation of the products of bacterial metabolism.

Some experiments performed with the assistance of Doctor Schechter on low calcium, which he had shown ('37) prolongs the life of the sea urchin egg, gave similar results.

RESPIRATION AND LIFE SPAN OF EGGS 133

RESPIRATION O F THE BACTERIA

The oxygen consumption of the bacteria was measured in a suspension of autoclaved, fragmented eggs. It is well known that the respiration of bacteria varies with the medium employed (Johnson, '37, for marine bacteria). The medium of autoclaved sea urchin eggs was employed in order to approxi- mate the conditions in the experiments with aging eggs. The bacterial suspension was prepared from plate washings and the oxygen consumption was followed in sea water as well as in the egg broth medium. Plate counts were made of samples taken from the vessels a t the end of the run. For the respiration in sea water the values 0.4 and 0.7 cu.mm. 0, per lo8 cells per hour were obtained in the two vessels that were run. For the oxygen consumption in the egg broth, the figures are 11.3 and 12.2. The sterile egg broth gave no measurable oxygen consumption. Waksman and Carey ('35) measured the oxygen consumption in stored sea water and obtained an average value of 1.7 cu.mm. per lo8 bacteria per hour (22°C.). John- son ('37) obtained for the average of twenty-four species measured separately 2.1 cu.mm. (25°C.) with a range of from 0.2 to 13.2 cu.rnm. for the different species. The addition of glucose gave a sevenfold increase on the average.

I n the experiments with aging eggs, the calculations of the bacterial respiration (assuming all of the excess respiration to be due to bacteria) shows enormous variations during the course of a run. It is usually much higher (about ten to fifty times) in the early part than in the later part of a run. We might, however, compare the maximum values with the value (11.8) obtained for the respiration in egg broth. In five experiments with aging eggs in which bacterial counts were made at three different intervals the maximum values obtained were 38.4, 10.3, 45.0, 33.2 and 42.2 cu.mm. per lo8 bacteria per hour. The average is then three times the value obtained in the egg broth and the highest value is almost four times.

From this comparison it might be concluded either that the excess respiration of the aging eggs was not entirely due to bacteria o r that the freshly disintegrating eggs provided more


readily oxidizable substrate than the medium of autoclaved egg broth.

RESPIRATION O F EGGS UNDER STERILE CONDITIONS

The procedure employed for obtaining a sterile suspension of Arbacia eggs was to remove the spines of the animal, wash it quickly in tap water, 95% alcohol and autoclaved sea water. Ripe animals are usually stimulated to shed by the treatment and the eggs are collected in autoclaved sea water. After four o r five washings with the sterile sea water the eggs are trans- ferred to the vessels, taking ordinary precautions to guard against contamination. Air contamination has generally been found to be almost negligible in the case of marine bacteria. However, in the laboratory it is necessary to guard against sea water spray. The sterility of the suspension in the vessels was tested by plating out at the end of a run. The vessels contained 7 cc. of fluid, and 1 cc. was plated out straight and in serial dilution. I n many instances, even when the original suspension of eggs showed no bacteria, they would later be present in the vessels. This may be due either to their having been missed in the original sample or to subsequent contamination. We shall refer to all supposedly sterile cultures as sterile whether or not they later showed bacterial growth.

When the respiration is measured under sterile conditions it is found to remain constant for prolonged periods while that of the non-sterile controls rises. Unfortunately, in none of our experiments did the eggs disintegrate while under sterile conditions. Figure 2 illustrates the results of an experiment with sterile and non-sterile eggs, the latter having been prepared by the addition of bacteria to a part of the sterile suspension. One pair of vessels was removed a t 14 hours, another pair a t 21 hours and the third ‘sterile’ vessel at 113 hours. The numbers of bacteria present are given at the corresponding points on the curves of figure 2. The non-sterile vessels show the usual rise in rate of oxygen consumption wit.h time. The sterile vessels give a constant rate for a prolonged period. At about 90 hours, however, the rate began to

EESPIRATION AND LIFE SPAN OF EGGS 135

rise in the remaining sterile vessel, reaching a maximum value about as high as that of the non-sterile vessels. The plate counts from this vessel showed that at the time of removal (113 hours) almost as many bacteria were present as in the non-sterile vessel removed at 21 hours. During the course of such a prolonged run it is necessary to introduce air into the vessel to readjust the manometer fluid, and this may have been

Fig. 2 Rate of oxygen consumption of unfertilized Arbaeia eggs under sterile and non-sterile conditions. The figures on the graph give the number of bacteria per cubic centimeter in the original egg suspensions and in the vessels removed at 14, 21 and 113 hours.

the source of the bacteria in spite of the precaution of intro- ducing the air through a drying tube. On the other hand, it is quite possible that the original suspension was not completely sterile even though the plates showed no bacteria.

The eggs from the vessels removed at 14 hours gave 95% cleavage in the case of the sterile suspension and 65% in the case of the non-sterile. At 21 hours 70% cleavage was obtained with the sterile eggs and no cleavage with the non- sterile, most of the latter being cytolyzed or disintegrated at

136 A. TYLER, F. RICCI AND N. H. HOROWITZ

this time. Of the eggs removed at 113 hours, 30% still appeared intact and 40% of these divided after insemination. The eggs in this vessel were examined from time to time with a low power microscope without removing the vessel from the manometer. As late as 90 hours, practically all of the eggs appeared intact.

Similar results were obtained in three other experiments of this sort. Whenever there was complete sterility or a relatively low number of bacteria the respiration remained at approximately the original value or showed a decrease in rate. Eggs from such vessels, removed even after the controls were completely disintegrated, mere intact and fertilizable. The supposedly sterile runs in which the eggs did finally cytolyze and disintegrate showed a high bacterial count as well as the rise in rate of oxygen consumption. No cases were obtained in which the eggs cytolyzed while the cultures remained sterile. This does not mean that eggs will not disintegrate under sterile conditions. However, it is difficult to obtain complete sterility in the heavy suspensions of eggs needed for the respiration runs, and even a single bacterium will give finally as much growth as the large number initially present in the non-sterile egg suspensions.

LENGTH O F LIFE UNDER STERILE CONDITIONS

To determine the length of life under sterile conditions the eggs were kept at room temperature (about 20.C.) in small Petri dishes sealed with vaselin. The eggs were removed directly from the ovary, washed a t least six times, and a known number (ten to twenty) placed in the Petri dishes which contained 4 cc. of the sterile sea water. When the dishes were opened 1 cc. of the sea water, containing usually one-fourth of the eggs, was plated out to test for sterility. The cultures termed sterile in this section showed no bacterial growth on the plates. For the non-sterile controls bacteria were added to the autoclaved sea water.

The length of life of the unfertilized egg was found to be considerably prolonged under sterile conditions. There is, of


course, great variation in the different cultures, but on the average the sterile cultures last about 10 days as compared with 2 days for the non-sterile. In one of the sterile cultures good eggs were obtained after 20 days, while with non-sterile cultures 3 days was the maximum.

Most of the data from three sets of experiments are given in table 1 in which is listed the number of eggs that cleaved upon insemination (C), that were uncleaved but apparently intact (I), and that were cytolyzed or disintegrated (D).

The difference between the sterile and non-sterile cultures is quite striking, amounting to a more than fivefold increase in the life span. The alcohol gives the usual prolongation in

TABLE 1 Dav? of

agzng Sterile Non-sterile C I D C I D

2 97 9 6 14 15 59 4 4 2 0 0 0 8 28 6 3 3 0 0 0 0 16 8 1 7 0 0 0 0 19 10 12 2 9 0 0 8 14 0 8 5 0 0 6 17 2 24 37 0 0 46 20 2 9 2 0 0 11

Sterile + 2 70 alcohol

C I D 81 15 2 18 10 2

8 0 13 0 6 11 1 4 7 0 6 6 0 0 61 0 2 15

Non-sterile + 2vo alcohol

C I D 37 45 5

7 4 12 1 6 18 0 0 16 0 5 11 0 0 16 0 0 38 0 0 12

the non-sterile cultures, but not in the case of the sterile cultures .

Distinction should of course be made between the length of fertilizable life and the total life span of the egg. It is, however, rather difficult to determine the actual time of death. In fact, after the loss of fertilizability we have no other very certain test of the viability of the unfertilized egg. For pur- poses of comparison, however, it is sufficient to apply some arbitrary criterion, such as the beginning of cytolysis. On this basis, the data show that the total life span, as well as the length of the fertilizable period, is extended by the sterile conditions. This does not mean that bacteria are directly re- sponsible for the death of the eggs, since it is quite evident that eggs do disintegrate under sterile conditions. It should also be pointed out that the 2% alcohol does not have any


bacteridical action since the same counts of viable bacteria are obtained from non-sterile sea water with and without the alcohol.

LENGTH O F LIFE IN BACTERIAL SUSPENSIONS

The addition of bacteria shortens somewhat the life span of the eggs, but the effect is not directly proportional to the amount of bacteria added. In four experiments, in which the eggs were placed in bacterial suspensions ranging up to about 100,000 times the control, the maximum shortening was about 30 to 40%. I n general, the eggs in both the bacterial suspensions and in the sea water controls remain intact and fertilizable up to about 36 hours. Then disintegration proceeds much more rapidly in the bacterial suspensions, so that by 45 to 50 hours they are completely disintegrated, whereas the same condition is not reached in the controls until 65 to 75 hours.

The presence of 2% alcohol extends the life span in the moderately heavy and weak bacterial suspensions as well as in the sea water controls. On the other hand, a considerable shortening is obtained in very heavy bacterial suspensions with 2% alcohol. For example, in a suspension containing roughly lo9 bacteria per cubic centimeter with 2% alcohol, the eggs were unfertilizable after 20 hours, whereas without the alcohol 85% cleavage was obtained a t that time, the eggs be- coming unfertilizable at 45 hours. It was noted that the eggs were decolorized in the heavy bacterial suspension with alcohol. The pH was therefore taken and it was found to have dropped to 4.7, while in the same bacterial suspensions without the alcohol it remained a t 8.1. The effect here is evidently due to acid production from the oxidation of the alcohol by the bacteria.

In these experiments with aging eggs, observations were also made on membrane elevation. Three types of behavior are in general noted: 1) normal membrane elevation, 2) tight membrane, 3) no membrane. After about 24 hours aging fertilization results in tight membranes and after about 36 hours

RESPIRATION A N D LIFE S P A N OF EGGS 139

there are no membranes formed. Neither the sterile conditions nor the presence of bacteria have any significant effect on the period during which normal or tight membranes are formed. Alcohol, however, extends this period to about double the control time.

DISCUSSION

It is apparent from the results that the rise in respiratory rate exhibited by aging unfertilized eggs can be accounted for by bacterial respiration. Along with the rise in respiration, there is a considerable growth of bacteria. When the rise is delayed as in 2% alcohol or in nearly sterile conditions, bacterial growth is also retarded. The rise is not directly proportional to the number of bacteria present, n w does the respiration of bacteria measured in egg broth quite reach the maximum values obtained in aging cultures of eggs on the assumption that the excess respiration is due to bacteria. But this may be due to differences in the culture conditions.

To definitely prove, however, that autolysis of the unfertilized egg is not accompanied by a rise in respiration, it would be necessary to have disintegration proceed under sterile conditions in the respiration vessels. This was not accomplished in the experiments. Supposedly sterile vessels that were run sufficiently long for the eggs to disintegrate showed a t the time of removal a high bacterial count as well as a rise in respiratory rate. Disintegration of the eggs can, of course, be produced mechanically and the respiration measured before there is any appreciable growth of bacteria. Warburg and Meyer- hof ( '12) found the respiration of unfertilized eggs that had been ground with sand to be less than that of the intact eggs. During the first hour it was about two-thirds of the control rate and by the third hour it sank to one-fourth. Warburg ( '14) found after destroying the unfertilized eggs by shaking an approximately 65% increase during the first half hour and likewise a subsequent falling off in rate. We have obtained similar results by freezing the eggs or treating them with distilled water. On the other hand, the increase in respiration


in the aging cultures of unfertilized eggs may be as much as 2000%. This would tend to support the view that the increase is due to bacteria. However, the reservation must be made that autolysis and mechanical cytolysis may not produce the same change in the egg.

If the conclusion is correct that the respiration remains constant or shows a drop in aging unfertilized eggs, then by comparison with fertilized eggs it appears that they die long before their reserve food material is oxidized. The fertilized sea urchin egg lives without feeding for about 2 weeks at room temperature and during most of this time its rate of oxygen consumption is about twenty to thirty times that of the unfertilized eggs. We should therefore expect the unfertilized egg to last for about 40 weeks instead of the maximum of 3 weeks o r the average of 1 week that we find. It may be concluded, then, that other processes than oxidation predominate in the autolysis of the unfertilized egg. This is in agreement with the general results of autolysis of various kinds of tissues (see review of Haehn, '36) in which the respiratory enzymes are to a large extent inactivated while hydrolytic enzymes are activated .

It is of some interest to consider whether sterile conditions and alcohol act similarly in prolonging the life span. The effect of the alcohol might conceivably be due to its antiseptic properties. Such was the interpretation offered by Gorham and Tower ( '02) for the action of KCN which Loeb and Lewis ('02) had shown would prolong the life of the unfertilized egg. This, they thought, was consistent with their finding that sterile conditions considerably prolong the fertilizable life of the eggs. Objections to their conclusions were subsequently raised by Loeb ('11) principally on the basis that anaerobiosis also prolongs the life of the egg. But for that contention to be valid, it would be necessary to show that anaerobiosis does not have any bactericidal action. In the case of alcohol, the concentrations employed in Whitaker 's ('37) and in our experiments are not bactericidal. The delayed bacterial growth in the egg suspensions with alcohol is


simply due to the slower disintegration of the eggs which fur- nishes the nutritive material f o r the bacteria. There is then a difference in the action of alcohol and of sterile conditions.

It is, of course, clear that the eggs do disintegrate under perfectly sterile conditions. That they die much sooner under non-sterile conditions means that bacteria (or the products of their metabolism) can attack the eggs. We may interpret the results then as follows. The unfertilized egg upon being placed in sea water begins to undergo disintegrative (autolytic) changes. After these changes have proceeded to a certain extent the eggs become susceptible to bacterial action, which speeds up their disintegration. In an ordinary sea water culture there are relatively few bacteria initially present, but as the first few eggs disintegrate material is made available for bacterial growth and so the disintegration proceeds with increasing rapidity. The assumption of an immune period explains why the addition of a heavy suspension of bacteria to fresh eggs does not cause immediate or very rapid death, but gives a relatively small shortening of the life span as compared with ordinary sea water cultures. The alcohol is, then, considered to act by retarding the autolytic changes and thus extending the immune period. That disintegrative changes are occurring during the period in which the egg is still fertilizable, we consider to be manifest by the loss of membrane forming capacity (in the sea urchin egg) as well as by the failure of eggs, fertilized toward the end of their span, to proceed very far in development. From our observations it appears that the time a t which the eggs become susceptible to bacterial attack coincides with the loss of membrane forming capacity. I n alcohol the eggs retain this capacity longer, but sterile conditions do not appear to influence this.

Of several interpretations for the action of alcohol and of dextrose, Whitaker considers most plausible that they serve as readily metabolizable nutrients. Since we failed to obtain extension of the life span with alcohol in sterile conditions, we should like to offer another interpretation. Marine bacteria are capable of oxidizing the alcohol to acid (probably


acetic) as we have seen particularly when heavy suspensions of bacteria were used. It is quite possible, then, that in ordinary non-sterile cultures, it is not the alcohol as such, but the slight amount of a weak acid produced from the alcohol, that extends the life span of the eggs. The action of dextrose could be similarly explained. Also in the case of KCN and of di- nitrophenol we are dealing with weak penetrating acids. Anaerobiosis and CO would give the same effect by causing the acid production within the cell.

SUMMARY

1. The rise in respiratory rate exhibited by aging unfertilized eggs is coincident both with loss of fertilizability (and incipient disintegration) of the eggs and bacterial growth in the vessels.

2. In 2% alcohol, which extends the life of the eggs, the respiratory rise is delayed, but there is also a corresponding delay in bacterial growth. The alcohol has no effect on the initial rate of respiration.

3. The respiration under sterile conditions remains low or even shows a slight drop as long as the vessels remain sterile. Eggs have been run for 4 days without exhibiting a rise in respiratory rate, whereas under ordinary conditions the rise begins at about 10 hours. 4. It appears that bacteria can account for the respiratory

rise, but it is not entirely excluded that autolysis of the eggs is accompanied by increased respiration, since in none of the respiration experiments was disintegration of the eggs obtained under sterile conditions.

5. Culturing eggs under sterile conditions extends their life span from an average of about 2 days to 10 days and from a maximum of 3 days to 20 days. Eggs do, however, disinte- gate under perfectly sterile conditions. Addition of large quantities of bacteria accelerates somewhat the disintegration of the eggs.

6. Alcohol in ordinary sea water or in dilute suspensions of bacteria approximately doubles the life of the unfertilized egg, but gives no prolongation under sterile conditions.

RESPIRATION AND LIFE SPAN O F EGGS 143

7. The results are taken to mean that bacteria contribute to the destruction of the eggs only after certain disintegration changes have occurred. The agents, such as alcohol, that prolong the life do not act bactericidally but by retarding these initial spontaneous changes on the part of the egg. Death under sterile conditions evidently oeeurs long before the oxidizable substrate is used up.

LITERATURE CITED

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pp. 172-182. HAEHN, H. 1936 Autolyse. Erg. d. Enzymforschung, Bd. 5, S. 117-158. JOHNSON, F. H. 1936 The oxygen uptake of marine bacteria. J. Bact., vol.

LOEB, J. 1911 Arch. Entw.-meeh., Bd. 31, S. 658-668.

Lorn, J,, AND W. H. LEWIS 1902 On the prolongation of the life of the unfertilized eggs of sea urchins by potassium cyanide. Am. J. Physiol., vol. 6, pp. 305-317.

RUNNSTROM, J. 1928 Struktur und Atmung bei der Entwicklungserregung des Seeigeleies. Acta Zool., vol. 9, pp. 445-449.

SCHECHTER, V. 1937 Calcium reductioii and the prolongation of life in the egg cells of Arbacia punctulata.

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The respiration and fertilizable life of Arbacia eggs under sterile and non-sterile conditions

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