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The effect of decreased and increased temperatures on fertilization
Experimental
174 THE
EFFECT
OF DECREASED
TEMPERATURES B. E. HAGSTROM The Wenner-Gren
Cell Research 16, 174-183 (1959)
AND
INCREASED
ON FERTILIZATION and BRITT
Institute for Experimental
Biology,
HAGSTROM University
of Stockholm, Sweden
Received April 26, 1958
DURING the development of the “fertilization rate method” (cf. 18, lo]) the influence on fertilization in sea urchins of the external conditions was also studied. The effect of variations in temperature seemed in the first place to be of interest. Bury [2] observed an increased susceptibility to polyspermy at lowered temperatures. The present writers have also observed that the development of the block to polyspermy is delayed upon exposure to low temperatures, which is obviously correlated with a slow propagation of the cortical reaction and a delayed or incomplete development of the hyaline layer (cf. IS]). Allen and Hagstriim [l] were also able to show that the cortical’reaction was stopped if the temperature of the inseminated egg suspension was rapidly increased to about 32°C. This interruption of the cortical reaction fertilized eggs” and an increased tendency to polyresulted in “partially spermy. In the present work the gametes were subjected to varying temperatures before fertilization. In other experiments insemination was also performed at temperatures below or above the temperature range normally occurring in the sea.
EXPERIMENTAL
The present investigation was carried out at Stazione Zoologica, Naples, using the species of sea urchins commonly found in the Gulf of Naples as material. Most of the experiments were made with the gametes from Paracentrotus lividus and Arbacia lixula. In about 20 experiments eggs and sperm from Psammechinus microtuberculatus and Sphaerechinus granularis were used. The experiments were made according to the fertilization rate method [S, IO] where the spermatozoa are killed by adding sodium laurylsulphate to the mixture of eggs and sperm. The method has been somewhat modified and a short description is therefore given here. The undiluted sperm from the testes is mixed with sea water to give a final concentration of about lo8 spermatozoa per ml. One ml of this stock suspension is mixed Experimental
Cell Research 16
The
effect
of
temperature
on
fe~ti~i~~ti0~
with 29 ml of sea water in a 400 ml beaker, whereupon 70 ml of egg suspension are added quickly. This results in an instantaneous and thorough mixing of eggs and sperm. Before the experiment an adequate number of 100 ml beakers are charged with P-I.5 ml of a solution of sodium laurylsulphate in sea water. At appropriate intervals of time (e.g. 5, 10, 15, 20 etc. seconds) after insemination 10 ml of the egg suspension are poured into the beakers containing laurylsulphate. The final concentration of laurylsulphate becomes 0.001 per cent, which immediately interrupts fertilization. If there is any substance present in the experiment which might act injurious on the eggs or on the further development of the larvae the beakers are filled up with filtered sea water 4-G minutes after insemination whereupon the eggs are allowed to settle and the supernatant is replaced by pure sea water. The percentage of fertilized eggs is easily counted after the first cleavages; as a rule 250 eggs are counted in every beaker. RESULTS The present results were obtained during March-June 1954-1957. More than 200 fertilization rate experiments were made and the concentration of sperm in the experiments was varied over a wide range between 106-S x 107 spermatozoa per ml. Experiments were made with eggs with intact jelly coats and with jellyfree eggs as well but the presence or absence of the jelly per se seemed not to influence the effect exerted upon fertilization by the increased or decreased temperature. Ilowever, the removal of the jelly always brought about a marked increase in the fertilization rate as was previously described [3, Z9 8, 141. In order to ensure that the desired temperature was attained instantaneously a small volume of egg suspension was mixed with a large volume of heated or cooled sea water; the volumes were adjusted to give a tinal temperature of e.g. 30°C. In experiments in which the sperm was subjected to high or low temperatures a test tube containing the so called “dry sperm” was kept in water of the temperature desired. In other experiments the concentrated sperm was suspended at the moment of insemination in cooled or heated sea water. ‘lhe temperatrue was kept constant throughout 30 minutes from insemination and the resulting fertilization was determined 1-2 hours after insemination. The percentage of polyspermic eggs, which is possible to determine with high accuracy before the third cell division, was also counted at the same time. The first series of experiments was arranged in order to study the elect of low and high temperatures on the fertilization process. Experimental
Cell Research LB
B. E. Hagstriim and &iii Hagsir6m
176
The normal temperature of the sea water in Naples ranged during the time of observation between 13.5” and 18.5%; as is indicated below this considerable increase in temperature might, in any case partly, be responsible for the increased rate of fertilization, which the present writers have observed towards the culmination of the spawning season. (Even a difference in temperature of 2°C gives fertilization rate curves which are distinctly separated from each other).
-1 o-----o o-.-.-.4
5
10
20
30
40
60
80
100
120
i i
180 SECONDS
2 3
1i
co
Fig. l.-Eggs and sperm from Arbacia Zixula; eggs with intact jelly coats. Concentration of sperm 8 x 10B/ml. 1, eggs inseminated at 15°C (control); 2, eggs inseminated at 5’C; 3, eggs inseminated at 22%. 5, 10, 20, 30 etc. seconds after insemination the sperm was killed by adding sodium laurylsulphate (cf. [lo]).
As already mentioned the temperature of the experiment was reached immediately before insemination by mixing cooled or heated sea water with the egg suspension. In temperatures below that of the sea the fertilization rate was always found to be lower than that of the control. The fertilization rate decreases with temperature and at + 3°C only a minor part of the eggs became fertilized. In temperatures about 5°C the number of fertilized eggs almost attains the normal value though there is an appreciable decrease in the fertilization rate (cf. Fig. 1). If fertilization is carried out at temperatures above that of the control there is a very evident increase in the rate of fertilization up to a certain level. This level is somewhat varying for the different species and Arbacia Iixula seems to resist increased temperatures better than does e.g. Psammechinus microtuberculatus, which observation is in accordance with the fact that Arbacia has its spawning season during the summer when the temperature Experimental
Cell Research 16
The effect of temperature on f~rt~lizati~~
177
of the sea has become increased. As a rule the upper limit for a positive response to increased temperatures lies about 3234°C. Above these temperatures the gametes are severely affected and above 40°C fertilization is hardly possible. The decreased or increased temperature at the moment of ~ertiI~zatio~ also influenced many morphological characters of the larvae. The elevation of the fertilization membrane was strikingly impaired in temperatures below that of the control. In temperatures up to 30-32°C the membranes were normal or even more elevated than in the control. At above 32°C the rnernb~a~~~ were more or less granular and adhering to the egg surface. The development of the hyaline layer was also effected in a similar way. If fertilization was carried out at low temperatures the hyaline layer became very thin; the formation of this layer was retarded as was also the elevation of the fertilization membrane. If the temperature was kept above that of the control the hyaline layer developed in a normal manner up to 30-32°C. f temperature was still more increased the appearance of the hyaline layer became ab rmal; many corticaI granules remained undissolved in the egg surface a yaline layer was rough. It was recently described that the cortical reaction may be stopped upon a short exposure of the inseminated eggs to a temperature of about 32°C [ B]. In the experiments of Allen and Hagstrom [1] the eggs were inseminated in sea water of normal temperature and the transfer to beated sea wat performed after the cortical reaction had already started. Thus th were subjected to the increased temperature in a shocklike manner. present experiments eggs and sperm interacted from the beginning in a of increased temperature; this difference might explain why the peree~t~~e of eggs showing interrupted cortical reaction was much lower in these Iatter owever, in some cases a stopping of the cortical reaction was lready mentioned the number of unfertilized eggs (i.e. e in which the sperm could not initiate any cortical reaction) increased with increasing temperature. As a consequence of the abnormal development of the hyaline layer e inseminated in sea water of temperatures considerably diverging from t of the control showed an increased incidence of polyspermy. The cleav and further development were also influenced in an abn The mechanism preventing polyspermy in sea urchins ted by the present writers [7, 8, 11, 121 and we arrived a the hyaline layer represents a barrier to polyspermy. The present work has 12 - 593701
Ex2werimenlal
Cell Resemch 1
B. E. Hagstriim and Britt Hagstr6m also produced strong evidence in support of this view. It was found that the injuries to the hyaline layer not only induced polyspermy but also effected abnormal cleavage and ensuing development. The formation of cell plates, which is a common occurrence after treatment with trypsin or Ca-free medium (cf. [ll, la]), is also a common phenomenon if fertilization is carried out at temperatures below + 10°C or above 32-34°C. The rate of the ensuing development is also influenced by the temperature at which fertilization is carried out. Though the temperature was only kept constant during 30 minutes from insemination (then the inseminated egg suspension was allowed to attain the temperature of the room) this treatment effected differences, which could be registered several hours later. Both low temperatures and temperatures above 32°C acted depressing on the rate of development and this effect was obviously correlated with the deficient formation of the hyaline layer, seen already within a minute or two after fertilization. In another series of experiments the gametes were only pretreated at immediately before the moment of decreased or increased temperatures; insemination the temperature of the gametes was returned to that of the control. Also in these experiments eggs with and without jelly coats were used but the presence or absence of the jelly seemed not to influence the effect of the temperature treatment applied. The eggs were allowed to settle on the bottom of a beaker and the supernatant sea water was sucked off. Ten ml of the dense egg suspension were then transferred to a beaker containing 90 ml of sea water (heated or cooled) and the temperature of the water was balanced to give the temperature of the experiment. The time of exposure to the decreased or increased temperature was varied from 2-30 minutes and the temperature ranged between 3” and 40°C. The beaker containing the eggs was &placed in a water bath and the temperature was kept constant during the period of treatment whereupon the supernatant sea water was sucked off and filtered sea water of the same temperature as that of the control was added. The control eggs were handled in the same way (except for the temperature treatment) and were subjected to the same changes of sea water. The results from these experiments were entirely different from those obtained in the experiments referred to above. Eggs stored for e.g. 5-10 minutes at 5°C showed a very marked raise in the fertilization rate; the same was found for eggs pretreated at 30°C for 10 minutes (cf. Fig. 2a). It was observed that a comparatively short treatment, i.e. 5-10 minutes, effected a more pronounced increase than did a pretreatment for 30 minutes. Experimental
Cell Research 16
179
The effect of temperature on fert~~i~~t~~~ Moreover, it was observed that pretreatment creased the fertilization rate a little more than The upper limit for a positive effect was with slight variations for the different species
of the eggs at 30°C usually indid pretreatment at 5” or 10°C. found to lie between 35-40°C tested. It was obvious that the
o------Q1 Q------c)
2
0 .. ... . .. ..0 3 *.-.-.a
5
10
15
20
4
40 SECONDS
Fig. 2a.
100 F p.“
80
:t: i 2E40 2 zs20
. ..’
/
,’
60
,” N -
c
-1
c-----Q2 O.........~
5
10
15
20
8 40
3
,I ‘; 4
Fig. 2 b. Fig. 2.-Eggs and sperm from Paracentrotus of sperm 5 x 10S/ml.
lividus;
eggs with
intact
jelly coats. Concentration
Fig. 2a.-1, Eggs inseminated at 18°C (control); 2, eggs pretreated for 3 minutes at 5’C; insemination at 18°C; 3, eggs pretreated for 3 minutes at 30°C; insemination at 18°C; 4, eggs pretreated for 10 minutes at 30°C; insemination at 18°C. Fig. 26.-Z, Control (cf. Fig. 2~); 2, the dry sperm was pretreated for 10 minutes at 5°C; insemination at 18°C; 3, the dry sperm was pretreated for 10 minutes at 3O’C; insemination at 18°C.
180
B. E. Hagsfrijm and Britf Hagsfriim
eggs did not stand the treatment at tion of high temperature and long the eggs and lowered the fertilization however, occasionally registered a 3 minutes at 40°C; this shows that valuable though a longer treatment
high temperatures very well; a combinatime of pretreatment produced injury to rate (cf. Fig. 3, curve no. 5). We have, positive response after pretreatment for a short shocklike pretreatment might be at the same temperature is harmful.
SECONDS
Fig. 3.-Eggs with jelly coats from Paracentrotus lividus. Concentration of sperm 106/ml. Insemination was carried out at 18%. 1, Control. Insemination at 18%; 2, eggs pretreated for 3 minutes at 5’C; 3, eggs pretreated for 3 minutes at 35’C; 4, eggs pretreated for 5 minutes at 35%; 5, eggs pretreated for 10 minutes at 35°C.
Cleavage and further development were generally favoured by pretreatment at decreased as well as at increased temperatures. After pretreatment at temperatures above 35°C there was, however, a tendency of the larvae to become pathological and to form cell plates. The incidence of polyspermy after pretreatment of the eggs at temperatures ranging between 5” and 35°C was very low and did not significantly deviate from that of the control. This is of particular interest because it demonstrates that the block to polyspermy is only influenced by e.g. heath if the increased temperature is applied during the propagation of the cortical reaction. Here it might also be inferred that cooling or heating of the eggs between 635°C .after that the cortical reaction was finished did not interfere with the block to polyspermy. In the last group of experiments the sperm was pretreated at different temperatures between 3” and 40°C. Experimental
Cell Research 16
The effect of temperature on ~ertiti~QtiQ~ Two types of experiments were made differing in the mode of wbicb t treatment at decreased or increased temperatures was applied. In one series of experiments the dry sperm was diluted in sea water of k desired temperature for l-10 minutes before insemination; 1 ml of this sto suspension was used for insemination. In another series the concentrated sperm from the testes (dry sperm) was kept in a water bath of the temperature desired. The concentrated sperm was then diluted in sea water of normal temperature immediately before insemination. The time of treatment was in this series l-30 minutes. In both types of experiments there was a considerable increase in tbe fertilization rate. However, the improvement was as a rule more obvious i eriments of the latter type. The increase in the fertilization rat cts an improvement of the fertilizing capacity of the sperm membrane elevation was also improved and became more uniform upon retreatment of the sperm. Fig. 2 b illustrates an experiment in whit ry sperm was cooled and heated respectively before insemination. As was already indicated [6] the quality of the sperm determines to a certain extent the quality and height of membrane elevation. This is proba also a reflection of the fact that the cortical reaction is more complete if the initiating sperm is in a good condition [ 171. In the present experiments it was observed that the hyaline layers became thicker and the membranes more highly elevated if pretreated sper used for insemination. Consequently the block to polyspermy was no tively influenced by pretreatment of the sperm. Also cleavage and development were found to be normal or even improved upon pretreatment of the sperm. Some experiments were also made in which pretreatment of sperm eggs were combined. The effect of these pretreatments were found to be a tive to a certain extent and the fertilization rate was increase reached by pretreatment of solely the eggs or the sperm.
DISCUSSION
The fertilization process is very complicated and our knowledge about the changes following the copulation of a spermatozoon and an egg is still fragmentary. It would therefore be premature to ascribe the effect registered upon beating or cooling of the gametes to one or another simple reaction wit the gamete. We know that enzymatic reactions are initiated upon fertilization and it may therefore be warranted to conclude that e.g. a moderate increase Experimental
Cdl Research Ph
182
B. E. Hagstriim and Britt Hagstrtim
in temperature would also increase the rate of these reactions. It still remains, however, to explain why also cooling of the gametes brings about an increased reactivity of the gametes. One of the present writers has previously studied the fertilization of so called underripe sea urchin eggs ([13], cf. also [17]). It was found that a storage of the eggs for 2-3 hours in sea water of normal temperature brought about an obvious increase in the fertilization rate. The present results show that the improvement of the egg material might also be achieved by a short heating or cooling of the egg suspension. Moreover, a similar improvement of the spermatozoon is also brought about by the same treatment of the sperm. This might be of interest from a practical point of view because it is possible within the course of a few minutes to bring up a heterogeneous or underripe material on a level of high fertilizability. Moreover, the results indicate that it is very important to keep the temperature constant and it is not advisable to expose the gametes to increased or decreased temperatures before or during the course of the experiment. The present work has clearly indicated that the block to polyspermy is related to the development of the hyaline layer. Previous experiments [l] showed that parts of the egg surface which are not covered by the cortical reaction are susceptible to refertilizing spermatozoa. It has been argued that the relatively high resistance to polyspermy, which is found to exist even in considerable concentrations of sperm must reside in an extremely rapid block to polyspermy, which is propagated in the egg surface within the course of a few seconds after sperm attachment (cf. [16]). The results of Allen and Hagstrijm (Cl], cf. also [4, 7, 8, 91) were entirely contradictory to this hypothesis [16]. It has, however, been claimed that this rapid block is reversed by heating the eggs [15]. However, unfertilized eggs which had been pretreated in heated sea water did not become polyspermic upon insemination. Moreover, it was obvious that it is just the very stopping of the cortical reaction (and consequently also the stopping of the formation of the hyaline layer) which makes the egg susceptible to polyspermy. The interuption of the cortical reaction is achieved if temperature is increased in a shocklike manner [l]; in the present experiments where insemination was carried out at increased temperature the incidence of polyspermy was low even at temperatures of 32-34°C. This obviously indicates that the cortical reaction is usually not stopped if the increased temperature is applied already at the moment of insemination. This result does not support the view of a hypothetical fast block which is abolished by heat.
Experimenfal
Cell Research 16
The effect of temperature on fert~~~~at~~~ SUMMARY The influence of decreased and increased temperatures on fert~l~zat~o~ 4n sea urchins was studied using the fertilization rate method. It was found that temperatures lower than that o e sea inhibited fertilization whereas fertilization was promoted by a sli ture. At temperatures above 32°C the fertilization rate was retarded. The gametes were also subjected to a short treatment at decreased or increased temperatures before insemination. In both types of experiments there was a considerable increase in the fertilization rate. The authors wish to express their sincere gratitude to Professor John Runnstrb;m for his kind interest in khe work. The work was supported hy a grant from “Statens Naturvetenskapliga Forskningsr&d” which is hereby acknowledged. REFERENCES 1. ALLEK, R. D. and WAGSTRGM, B. E., Exptl. Cell Research 9, 157 (1955). 2. BURY, J., Arch. E~twicklungsmech. Organ. 36, 537 (1913). B. E., Exptl. Cell Research 10, 24 (1956). 3. WAGSTR~M, 4. I_ 5. I_
6. 7. 8. 9. 10. 11. 12. 13. 14.
15. 16. 17.
ibid.
10, 740 (1956).
ibid. 11, 306 (1956). __ ibid. 11, 507 (1956). __ Arkiv Zool. 10, 307 (1956). __ The Role of the Jelly Coat and the Block to Urchins. Dissertation, Uppsala, 1956. __ Exptl. CeZZResearch, 16, 184 (1959). HAGSTR~M, B. E. and NAGSTR~M, BRITT, Exptl. Cell __ ibid. 6, 491 (1954). __ ibid. 6, 532 (1954). HAGSTRBM, B., ibid. 9, 313 (1955). HAGSTR~M, BRITT and HAGSTRGX, B.E., ArkivZooZ. METZ, C. B., Physiological Triggers, p, 17, 1956. ROTESGMLD, LORD, Fertilization, Methuen, London, RUNSSTRSM~ J., Advances in Enzymol. 9, 241 (1949).
Polyspermy
in the Fertilization
of Sea
Research
7, 579 (19551. 1956.
Experimental
Gel! Research I.6
174 THE
EFFECT
OF DECREASED
TEMPERATURES B. E. HAGSTROM The Wenner-Gren
Cell Research 16, 174-183 (1959)
AND
INCREASED
ON FERTILIZATION and BRITT
Institute for Experimental
Biology,
HAGSTROM University
of Stockholm, Sweden
Received April 26, 1958
DURING the development of the “fertilization rate method” (cf. 18, lo]) the influence on fertilization in sea urchins of the external conditions was also studied. The effect of variations in temperature seemed in the first place to be of interest. Bury [2] observed an increased susceptibility to polyspermy at lowered temperatures. The present writers have also observed that the development of the block to polyspermy is delayed upon exposure to low temperatures, which is obviously correlated with a slow propagation of the cortical reaction and a delayed or incomplete development of the hyaline layer (cf. IS]). Allen and Hagstriim [l] were also able to show that the cortical’reaction was stopped if the temperature of the inseminated egg suspension was rapidly increased to about 32°C. This interruption of the cortical reaction fertilized eggs” and an increased tendency to polyresulted in “partially spermy. In the present work the gametes were subjected to varying temperatures before fertilization. In other experiments insemination was also performed at temperatures below or above the temperature range normally occurring in the sea.
EXPERIMENTAL
The present investigation was carried out at Stazione Zoologica, Naples, using the species of sea urchins commonly found in the Gulf of Naples as material. Most of the experiments were made with the gametes from Paracentrotus lividus and Arbacia lixula. In about 20 experiments eggs and sperm from Psammechinus microtuberculatus and Sphaerechinus granularis were used. The experiments were made according to the fertilization rate method [S, IO] where the spermatozoa are killed by adding sodium laurylsulphate to the mixture of eggs and sperm. The method has been somewhat modified and a short description is therefore given here. The undiluted sperm from the testes is mixed with sea water to give a final concentration of about lo8 spermatozoa per ml. One ml of this stock suspension is mixed Experimental
Cell Research 16
The
effect
of
temperature
on
fe~ti~i~~ti0~
with 29 ml of sea water in a 400 ml beaker, whereupon 70 ml of egg suspension are added quickly. This results in an instantaneous and thorough mixing of eggs and sperm. Before the experiment an adequate number of 100 ml beakers are charged with P-I.5 ml of a solution of sodium laurylsulphate in sea water. At appropriate intervals of time (e.g. 5, 10, 15, 20 etc. seconds) after insemination 10 ml of the egg suspension are poured into the beakers containing laurylsulphate. The final concentration of laurylsulphate becomes 0.001 per cent, which immediately interrupts fertilization. If there is any substance present in the experiment which might act injurious on the eggs or on the further development of the larvae the beakers are filled up with filtered sea water 4-G minutes after insemination whereupon the eggs are allowed to settle and the supernatant is replaced by pure sea water. The percentage of fertilized eggs is easily counted after the first cleavages; as a rule 250 eggs are counted in every beaker. RESULTS The present results were obtained during March-June 1954-1957. More than 200 fertilization rate experiments were made and the concentration of sperm in the experiments was varied over a wide range between 106-S x 107 spermatozoa per ml. Experiments were made with eggs with intact jelly coats and with jellyfree eggs as well but the presence or absence of the jelly per se seemed not to influence the effect exerted upon fertilization by the increased or decreased temperature. Ilowever, the removal of the jelly always brought about a marked increase in the fertilization rate as was previously described [3, Z9 8, 141. In order to ensure that the desired temperature was attained instantaneously a small volume of egg suspension was mixed with a large volume of heated or cooled sea water; the volumes were adjusted to give a tinal temperature of e.g. 30°C. In experiments in which the sperm was subjected to high or low temperatures a test tube containing the so called “dry sperm” was kept in water of the temperature desired. In other experiments the concentrated sperm was suspended at the moment of insemination in cooled or heated sea water. ‘lhe temperatrue was kept constant throughout 30 minutes from insemination and the resulting fertilization was determined 1-2 hours after insemination. The percentage of polyspermic eggs, which is possible to determine with high accuracy before the third cell division, was also counted at the same time. The first series of experiments was arranged in order to study the elect of low and high temperatures on the fertilization process. Experimental
Cell Research LB
B. E. Hagstriim and &iii Hagsir6m
176
The normal temperature of the sea water in Naples ranged during the time of observation between 13.5” and 18.5%; as is indicated below this considerable increase in temperature might, in any case partly, be responsible for the increased rate of fertilization, which the present writers have observed towards the culmination of the spawning season. (Even a difference in temperature of 2°C gives fertilization rate curves which are distinctly separated from each other).
-1 o-----o o-.-.-.4
5
10
20
30
40
60
80
100
120
i i
180 SECONDS
2 3
1i
co
Fig. l.-Eggs and sperm from Arbacia Zixula; eggs with intact jelly coats. Concentration of sperm 8 x 10B/ml. 1, eggs inseminated at 15°C (control); 2, eggs inseminated at 5’C; 3, eggs inseminated at 22%. 5, 10, 20, 30 etc. seconds after insemination the sperm was killed by adding sodium laurylsulphate (cf. [lo]).
As already mentioned the temperature of the experiment was reached immediately before insemination by mixing cooled or heated sea water with the egg suspension. In temperatures below that of the sea the fertilization rate was always found to be lower than that of the control. The fertilization rate decreases with temperature and at + 3°C only a minor part of the eggs became fertilized. In temperatures about 5°C the number of fertilized eggs almost attains the normal value though there is an appreciable decrease in the fertilization rate (cf. Fig. 1). If fertilization is carried out at temperatures above that of the control there is a very evident increase in the rate of fertilization up to a certain level. This level is somewhat varying for the different species and Arbacia Iixula seems to resist increased temperatures better than does e.g. Psammechinus microtuberculatus, which observation is in accordance with the fact that Arbacia has its spawning season during the summer when the temperature Experimental
Cell Research 16
The effect of temperature on f~rt~lizati~~
177
of the sea has become increased. As a rule the upper limit for a positive response to increased temperatures lies about 3234°C. Above these temperatures the gametes are severely affected and above 40°C fertilization is hardly possible. The decreased or increased temperature at the moment of ~ertiI~zatio~ also influenced many morphological characters of the larvae. The elevation of the fertilization membrane was strikingly impaired in temperatures below that of the control. In temperatures up to 30-32°C the membranes were normal or even more elevated than in the control. At above 32°C the rnernb~a~~~ were more or less granular and adhering to the egg surface. The development of the hyaline layer was also effected in a similar way. If fertilization was carried out at low temperatures the hyaline layer became very thin; the formation of this layer was retarded as was also the elevation of the fertilization membrane. If the temperature was kept above that of the control the hyaline layer developed in a normal manner up to 30-32°C. f temperature was still more increased the appearance of the hyaline layer became ab rmal; many corticaI granules remained undissolved in the egg surface a yaline layer was rough. It was recently described that the cortical reaction may be stopped upon a short exposure of the inseminated eggs to a temperature of about 32°C [ B]. In the experiments of Allen and Hagstrom [1] the eggs were inseminated in sea water of normal temperature and the transfer to beated sea wat performed after the cortical reaction had already started. Thus th were subjected to the increased temperature in a shocklike manner. present experiments eggs and sperm interacted from the beginning in a of increased temperature; this difference might explain why the peree~t~~e of eggs showing interrupted cortical reaction was much lower in these Iatter owever, in some cases a stopping of the cortical reaction was lready mentioned the number of unfertilized eggs (i.e. e in which the sperm could not initiate any cortical reaction) increased with increasing temperature. As a consequence of the abnormal development of the hyaline layer e inseminated in sea water of temperatures considerably diverging from t of the control showed an increased incidence of polyspermy. The cleav and further development were also influenced in an abn The mechanism preventing polyspermy in sea urchins ted by the present writers [7, 8, 11, 121 and we arrived a the hyaline layer represents a barrier to polyspermy. The present work has 12 - 593701
Ex2werimenlal
Cell Resemch 1
B. E. Hagstriim and Britt Hagstr6m also produced strong evidence in support of this view. It was found that the injuries to the hyaline layer not only induced polyspermy but also effected abnormal cleavage and ensuing development. The formation of cell plates, which is a common occurrence after treatment with trypsin or Ca-free medium (cf. [ll, la]), is also a common phenomenon if fertilization is carried out at temperatures below + 10°C or above 32-34°C. The rate of the ensuing development is also influenced by the temperature at which fertilization is carried out. Though the temperature was only kept constant during 30 minutes from insemination (then the inseminated egg suspension was allowed to attain the temperature of the room) this treatment effected differences, which could be registered several hours later. Both low temperatures and temperatures above 32°C acted depressing on the rate of development and this effect was obviously correlated with the deficient formation of the hyaline layer, seen already within a minute or two after fertilization. In another series of experiments the gametes were only pretreated at immediately before the moment of decreased or increased temperatures; insemination the temperature of the gametes was returned to that of the control. Also in these experiments eggs with and without jelly coats were used but the presence or absence of the jelly seemed not to influence the effect of the temperature treatment applied. The eggs were allowed to settle on the bottom of a beaker and the supernatant sea water was sucked off. Ten ml of the dense egg suspension were then transferred to a beaker containing 90 ml of sea water (heated or cooled) and the temperature of the water was balanced to give the temperature of the experiment. The time of exposure to the decreased or increased temperature was varied from 2-30 minutes and the temperature ranged between 3” and 40°C. The beaker containing the eggs was &placed in a water bath and the temperature was kept constant during the period of treatment whereupon the supernatant sea water was sucked off and filtered sea water of the same temperature as that of the control was added. The control eggs were handled in the same way (except for the temperature treatment) and were subjected to the same changes of sea water. The results from these experiments were entirely different from those obtained in the experiments referred to above. Eggs stored for e.g. 5-10 minutes at 5°C showed a very marked raise in the fertilization rate; the same was found for eggs pretreated at 30°C for 10 minutes (cf. Fig. 2a). It was observed that a comparatively short treatment, i.e. 5-10 minutes, effected a more pronounced increase than did a pretreatment for 30 minutes. Experimental
Cell Research 16
179
The effect of temperature on fert~~i~~t~~~ Moreover, it was observed that pretreatment creased the fertilization rate a little more than The upper limit for a positive effect was with slight variations for the different species
of the eggs at 30°C usually indid pretreatment at 5” or 10°C. found to lie between 35-40°C tested. It was obvious that the
o------Q1 Q------c)
2
0 .. ... . .. ..0 3 *.-.-.a
5
10
15
20
4
40 SECONDS
Fig. 2a.
100 F p.“
80
:t: i 2E40 2 zs20
. ..’
/
,’
60
,” N -
c
-1
c-----Q2 O.........~
5
10
15
20
8 40
3
,I ‘; 4
Fig. 2 b. Fig. 2.-Eggs and sperm from Paracentrotus of sperm 5 x 10S/ml.
lividus;
eggs with
intact
jelly coats. Concentration
Fig. 2a.-1, Eggs inseminated at 18°C (control); 2, eggs pretreated for 3 minutes at 5’C; insemination at 18°C; 3, eggs pretreated for 3 minutes at 30°C; insemination at 18°C; 4, eggs pretreated for 10 minutes at 30°C; insemination at 18°C. Fig. 26.-Z, Control (cf. Fig. 2~); 2, the dry sperm was pretreated for 10 minutes at 5°C; insemination at 18°C; 3, the dry sperm was pretreated for 10 minutes at 3O’C; insemination at 18°C.
180
B. E. Hagsfrijm and Britf Hagsfriim
eggs did not stand the treatment at tion of high temperature and long the eggs and lowered the fertilization however, occasionally registered a 3 minutes at 40°C; this shows that valuable though a longer treatment
high temperatures very well; a combinatime of pretreatment produced injury to rate (cf. Fig. 3, curve no. 5). We have, positive response after pretreatment for a short shocklike pretreatment might be at the same temperature is harmful.
SECONDS
Fig. 3.-Eggs with jelly coats from Paracentrotus lividus. Concentration of sperm 106/ml. Insemination was carried out at 18%. 1, Control. Insemination at 18%; 2, eggs pretreated for 3 minutes at 5’C; 3, eggs pretreated for 3 minutes at 35’C; 4, eggs pretreated for 5 minutes at 35%; 5, eggs pretreated for 10 minutes at 35°C.
Cleavage and further development were generally favoured by pretreatment at decreased as well as at increased temperatures. After pretreatment at temperatures above 35°C there was, however, a tendency of the larvae to become pathological and to form cell plates. The incidence of polyspermy after pretreatment of the eggs at temperatures ranging between 5” and 35°C was very low and did not significantly deviate from that of the control. This is of particular interest because it demonstrates that the block to polyspermy is only influenced by e.g. heath if the increased temperature is applied during the propagation of the cortical reaction. Here it might also be inferred that cooling or heating of the eggs between 635°C .after that the cortical reaction was finished did not interfere with the block to polyspermy. In the last group of experiments the sperm was pretreated at different temperatures between 3” and 40°C. Experimental
Cell Research 16
The effect of temperature on ~ertiti~QtiQ~ Two types of experiments were made differing in the mode of wbicb t treatment at decreased or increased temperatures was applied. In one series of experiments the dry sperm was diluted in sea water of k desired temperature for l-10 minutes before insemination; 1 ml of this sto suspension was used for insemination. In another series the concentrated sperm from the testes (dry sperm) was kept in a water bath of the temperature desired. The concentrated sperm was then diluted in sea water of normal temperature immediately before insemination. The time of treatment was in this series l-30 minutes. In both types of experiments there was a considerable increase in tbe fertilization rate. However, the improvement was as a rule more obvious i eriments of the latter type. The increase in the fertilization rat cts an improvement of the fertilizing capacity of the sperm membrane elevation was also improved and became more uniform upon retreatment of the sperm. Fig. 2 b illustrates an experiment in whit ry sperm was cooled and heated respectively before insemination. As was already indicated [6] the quality of the sperm determines to a certain extent the quality and height of membrane elevation. This is proba also a reflection of the fact that the cortical reaction is more complete if the initiating sperm is in a good condition [ 171. In the present experiments it was observed that the hyaline layers became thicker and the membranes more highly elevated if pretreated sper used for insemination. Consequently the block to polyspermy was no tively influenced by pretreatment of the sperm. Also cleavage and development were found to be normal or even improved upon pretreatment of the sperm. Some experiments were also made in which pretreatment of sperm eggs were combined. The effect of these pretreatments were found to be a tive to a certain extent and the fertilization rate was increase reached by pretreatment of solely the eggs or the sperm.
DISCUSSION
The fertilization process is very complicated and our knowledge about the changes following the copulation of a spermatozoon and an egg is still fragmentary. It would therefore be premature to ascribe the effect registered upon beating or cooling of the gametes to one or another simple reaction wit the gamete. We know that enzymatic reactions are initiated upon fertilization and it may therefore be warranted to conclude that e.g. a moderate increase Experimental
Cdl Research Ph
182
B. E. Hagstriim and Britt Hagstrtim
in temperature would also increase the rate of these reactions. It still remains, however, to explain why also cooling of the gametes brings about an increased reactivity of the gametes. One of the present writers has previously studied the fertilization of so called underripe sea urchin eggs ([13], cf. also [17]). It was found that a storage of the eggs for 2-3 hours in sea water of normal temperature brought about an obvious increase in the fertilization rate. The present results show that the improvement of the egg material might also be achieved by a short heating or cooling of the egg suspension. Moreover, a similar improvement of the spermatozoon is also brought about by the same treatment of the sperm. This might be of interest from a practical point of view because it is possible within the course of a few minutes to bring up a heterogeneous or underripe material on a level of high fertilizability. Moreover, the results indicate that it is very important to keep the temperature constant and it is not advisable to expose the gametes to increased or decreased temperatures before or during the course of the experiment. The present work has clearly indicated that the block to polyspermy is related to the development of the hyaline layer. Previous experiments [l] showed that parts of the egg surface which are not covered by the cortical reaction are susceptible to refertilizing spermatozoa. It has been argued that the relatively high resistance to polyspermy, which is found to exist even in considerable concentrations of sperm must reside in an extremely rapid block to polyspermy, which is propagated in the egg surface within the course of a few seconds after sperm attachment (cf. [16]). The results of Allen and Hagstrijm (Cl], cf. also [4, 7, 8, 91) were entirely contradictory to this hypothesis [16]. It has, however, been claimed that this rapid block is reversed by heating the eggs [15]. However, unfertilized eggs which had been pretreated in heated sea water did not become polyspermic upon insemination. Moreover, it was obvious that it is just the very stopping of the cortical reaction (and consequently also the stopping of the formation of the hyaline layer) which makes the egg susceptible to polyspermy. The interuption of the cortical reaction is achieved if temperature is increased in a shocklike manner [l]; in the present experiments where insemination was carried out at increased temperature the incidence of polyspermy was low even at temperatures of 32-34°C. This obviously indicates that the cortical reaction is usually not stopped if the increased temperature is applied already at the moment of insemination. This result does not support the view of a hypothetical fast block which is abolished by heat.
Experimenfal
Cell Research 16
The effect of temperature on fert~~~~at~~~ SUMMARY The influence of decreased and increased temperatures on fert~l~zat~o~ 4n sea urchins was studied using the fertilization rate method. It was found that temperatures lower than that o e sea inhibited fertilization whereas fertilization was promoted by a sli ture. At temperatures above 32°C the fertilization rate was retarded. The gametes were also subjected to a short treatment at decreased or increased temperatures before insemination. In both types of experiments there was a considerable increase in the fertilization rate. The authors wish to express their sincere gratitude to Professor John Runnstrb;m for his kind interest in khe work. The work was supported hy a grant from “Statens Naturvetenskapliga Forskningsr&d” which is hereby acknowledged. REFERENCES 1. ALLEK, R. D. and WAGSTRGM, B. E., Exptl. Cell Research 9, 157 (1955). 2. BURY, J., Arch. E~twicklungsmech. Organ. 36, 537 (1913). B. E., Exptl. Cell Research 10, 24 (1956). 3. WAGSTR~M, 4. I_ 5. I_
6. 7. 8. 9. 10. 11. 12. 13. 14.
15. 16. 17.
ibid.
10, 740 (1956).
ibid. 11, 306 (1956). __ ibid. 11, 507 (1956). __ Arkiv Zool. 10, 307 (1956). __ The Role of the Jelly Coat and the Block to Urchins. Dissertation, Uppsala, 1956. __ Exptl. CeZZResearch, 16, 184 (1959). HAGSTR~M, B. E. and NAGSTR~M, BRITT, Exptl. Cell __ ibid. 6, 491 (1954). __ ibid. 6, 532 (1954). HAGSTRBM, B., ibid. 9, 313 (1955). HAGSTR~M, BRITT and HAGSTRGX, B.E., ArkivZooZ. METZ, C. B., Physiological Triggers, p, 17, 1956. ROTESGMLD, LORD, Fertilization, Methuen, London, RUNSSTRSM~ J., Advances in Enzymol. 9, 241 (1949).
Polyspermy
in the Fertilization
of Sea
Research
7, 579 (19551. 1956.
Experimental
Gel! Research I.6