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The effect of oxidized lipoic acid on developing echinoderm embryos
66
Experimental
THE
EFFECT DEVELOPING NANCY
OF
OXIDIZED
LIPOIC
ECHINODERM WOLFSON
and DOROTHY
Department of Physiology, Mt. Department of Biology, Randolph-Macon Received
Cell Research
ACID
38, 66- 74 (1965)
ON
EMBRYOS S. FRY
Holyoke College S. Ha&y, Mass., and Woman’s College Lynchburg, Va., U.S.A. June 9, 1964
IN earlier studies we observed that homogenates of sea urchin eggs contained a cleavage inhibitor whose inhibitory action was intensified in the presence of sulfhydryl reagents [7, 81. In attempting to identify the cleavage inhibitor, we became interested in various naturally occurring sulfhydryl and disulfide compounds, and their effect on mitosis. Lipoic acid in its oxidized form is a cyclic disulfide well known for its role in oxidative decarboxylations. Recently it has been studied in a different context, that of its effect on morphogenetic and regenerative processes [3, 4, 5, 61. To distinguish between possible effects on cell division and on differentiation we have applied lipoic acid to developing echinoderm eggs whose early cleavages can be readily observed and timed. The experiments described indicate that low concentrations of oxidized lipoic acid have only slight effect on early cleavage but produce a marked inhibition of normal derelopment in sand dollar and sea urchin embryos. METHODS The observations were made on eggs of the sand dollar, Echinarachnius parnla, and of the sea urchin, Arbacia punctulata. The sand dollars were collected in Maine, at the Mount Desert Island Biological Laboratory, and the sea urchins in Connecticut and North Carolina, at the Marine Research Laboratory of the University of Connecticut, and the Duke University Marine Laboratory. The observations on Echinarachnius were made in July, and those on Arbacia, for the most part, in early spring. The lipoic acid was supplied in its oxidized form by General Biochemicals, Chagrin Falls, Ohio. A solution of the slightly soluble acid was prepared by converting it to the sodium salt with NaOH and neutralizing with HCl. A stock solution contained 2000 pg lipoic acid per ml in 0.04 M NaOH, 0.04 111 HCl, at pH 8, and was kept refrigerated until discarded after one week. 1 Present Experimental
address:
Department
Cell Research
38
of Zoology,
McGill
University,
Montreal,
Canada.
Lip&
acid on echinoderm
67
embryos
RESULTS
Experiments with Echinarachnius parma.-The eggs of the sand dollar, E. parma, are large and transparent, and the morphological transitions are much easier to follow than in the smaller, pigmented eggs of Arbacia. Shortly after fertilization the eggs were added to sea water containing various concentrations of lipoic acid, from lO-’ to 10~~ M. In these concentrations embryos developed normally up to the hatching blastula stage. The control embryos proceeded through hatching, gastrulation and pluteus formation while the embryos exposed to the lipoic acid solution remained as blastulae, spinning actively within their fertilization membranes. The inhibited embryos remained intact, often spinning for one or two days after their counterparts in sea water had formed long-armed plutei. Total and consistent blocking of this type was obtained at concentrations of 10P5 M. Some embryos were blocked at 1OF M and fewer were blocked at lo-’ M. Embryos exposed from fertilization through blastula stage to 1O-5 M lipoic. acid, and then removed to fresh sea water, remained in their inhibited state. The blocking was irreversible at that concentration for that duration of exposure. We observed little delay in cleavage rate in eggs developing in concentrations up to 200 ,ug lipoic acid (lop3 M). By counting eggs cleaved at several intervals, and by plotting these counts against time, rate curves are obtained and a very accurate time of 50 per cent cleavage found for controls and experimentals. The results of one such experiment to observe the effect of three concentrations of lipoic acid on the rate of early cleavage are shown in Table I. TABLE
I. Effect
lipoic acid on cleavage rate (Echinarachnius
of
parma).
The time at which 50 per cent of the eggs in any dish completed their second and third cleavages is shown in min after fertilization, for controls and experimental embryos exposed to three concentrations of lipoic acid (13°C). The delay accumulated by the experimental embryos at the end of the third cleavage is the difference in min as compared with controls. Concentration of lipoic acid kidml
0 4 40 200
Time of 50 y0 cleavage, Cleavage II Cleavage 160 161 164
213 220 220 216
min III
Delay at III
7 7 3
Experimental
Cell Research
38
68
Nancy
Wolfson
and Dorothy
S. Fry
It is apparent that very little delay was produced during the first three cleavages by any of the three concentrations of lipoic acid. Even concentrations of 200 ,ug/ml produced a delay of no more than 3 min by the end of third cleavage. The measurements on cleavage rates confirmed observations during the course of other experiments in which controls and experimentals did not vary in rate of development until after the blastula stage. Having found that lipoic acid at lo- 5 M had no discernible effect on cleavage rate or form but blocked further development at the spinning blastula stage, we next tried to find whether some stages of early development were especially sensitive to the inhibitory action of the reagent. Eggs were fertilized and exposed to 4 pg/ml of lipoic acid in sea water within 15 min after fertilization. Aliquots of eggs were removed from the lipoic-sea water at intervals, washed and placed in fresh sea water. Eggs removed from lipoicsea water at any time after completion of second cleavage generally showed characteristic abnormalities of development. Eggs removed before second cleavage usually did not show many such abnormalities. The duration of the exposure, from 15 min after fertilization through second cleavage, seemed a rough minimum exposure for obtaining a morphological effect, (about 2 hr at 15’C). We used the 2 hr interval for “pulse” exposures distributed over the developmental period from fertilization to gastrulation. Several abnormalities characterize the later development of embryos that have undergone short-term exposure to lipoic acid. These include (I) a delay in gastrulation, (2) disorganized mesenchymal proliferation into the blastocoel, (3) frequent occurrence of exogastrulae, and (4) abnormalities in the arms of individuals reaching the pluteus stage. We chose the appearance of exogastrulae as an easily recognizable criterion for assessing the effects of lipoic acid on development (Fig. 1 a). Two exogastrulae are shown in the figure. The embryo on the left shows well defined constrictions in the proximal region of the endoderm. The distal regions of the everted tube are less well defined, terminating in loose clusters of cells which tend to adhere to clusters on other exogastrulae, as shown in the photograph. The percentage of exogastrulae from samples of eggs exposed for 2 hr to lipoic acid at various times during development was determined for several egg batches. The results are tabulated in Fig. 2. Although the percentages Fig. l.-Characteristic abnormalities to lipoic acid for 2 hr during early lipoic acid for 2 hr, beginning shortly exposed to 100 ,ug/ml lipoic acid for Microscopic magnification x 200 for Experimental
Cell
Research
38
resulting from exposure of developing echinoderm embryos development. (a) Echinarachnius parma exposed to 4 pg/ml after fertilization. Age: 2 days at 15’C. (b) Arbacia punctulata 2 hr, beginning 4 hr after fertilization. Age: 4 days at 2O’C. both species.
Lipoic acid on echinoderm
embryos
Experimental
Cell
70
Nancy
Wolfson
and Dorothy
S. Fry
are too variable to distinguish among the sensitivities of the first four exposure periods, there is no question of the abrupt drop-off in sensitivity that occurs if the exposure to lipoic acid is made after the eighth hr of development. It is also clear from Fig. 2 that embryos exposed during early cleavages are sensitive to the adverse action of lipoic acid on their subsequent development, even though, as we have shown above, their cleavage rate was unaffected. Finally, we can note that some embryos are affected irreversibly by the lipoic acid, no matter which early developmental stage is exposed. Experiments with Arbacia punctulata.-Eggs of the sea urchin, A. punctulatu, were fertilized and lo-15 min later were transferred to sea water with TABLE
II. Effect
of tipoic acid on cleavage rate (Arbacia
punctulata).
The time at which 50 per cent of the eggs in any dish completed their first, second and third cleavages is shown in min after fertilization, for controls and experimental embryos exposed to three concentrations of lipoic acid (23°C). The delay accumulated by the experimental embryos at the end of the third cleavage is the difference in min as compared with controls. Eggs exposed to 100 pg/ml lipoic acid never achieved 50 per cent cleavage but began cleaving at the approximate time shown in parentheses. Concentration of lipoic acid &ml
Cleavage 67 68 68 70
0
4 40 100
Time
of 50 % cleavage,
min
I
II
III
108 110 118 (began 126)
142 146 158 (began 168)
Delay
at III
4 10 (approx.
26)
one of three concentrations of lipoic acid: 4, 40 or 100 rug/ml. At 4 ,ug/ml the embryos were slightly delayed in their development but eventually tormed normal plutei. At 40 pg/ml the embryos did not form plutei, and most of them stopped developing at, or before the blastula stage. The blocking of development was not as clearcut in Arbacia as in Echinarachnius. Many of the inhibited Arbacia embryos were irregular in form, some were unhatched and others were disintegrating within a few hours after exposure to the lipoic acid solution. At the highest concentration tested, 100 pg/ml, the embryos disintegrated before reaching blastula stage. At this concentration the deleterious effect of the reagent was noted by the time of second cleavage. The inhibition of early cleavage was more apparent in Arbacia than Echinarachnius (Table II). The delay in cleavage resulting from the lower c,oncentrations of lipoic acid was small but with 100 ,ug/ml, many embryos Experimental
Cell
Research
38
Lipoic
acid on echinoderm
71
embryos
had their cleavages blocked from the third cleavage on. For the experiment in Table II, at 100 pg/ml, nearly all the eggs completed first cleavage but less than 30 per cent of the embryos cleaved more than once. The time reported is that when the eggs began to cleave, and since fewer than 30 per Developmental
Hours 0
I
2
3
4
5
6
Periods
Expt. I.
of ter 7
46
fertilization
8
9
IO
of exposure
Percentage I2
stages
51
12
to lipoic
acid
I3
I4
I5
I6
I7
I8
I9
exogastrulae
75
2.
5
3.
0
4.
0
5.
-22
ave.
II
8
21
30
5
51 2
d!.iO~~~
23
37
0
0
0
0
0
0
0
0
0
0
0
0
--
26
0
I
0
Fig. 2.-The stages drawn in the top row of diagrams are: fertilized egg, 4-cell, l&cell, early blastula, mid-blastula, spinning blastula, late blastula, early gastrula. They are placed according to their occurrence in time after fertilization at 15°C. The duration and time of exposure of the embryos to 4 pg/ml lipoic acid are indicated by horizontal bars. A different sample from a single batch of fertilized eggs was exposed for each treatment in a given experiment. The percentage of exogastrulae resulting from each treatment is tabulated under the appropriate exposure period (Echinarachnius parma.)
cent completed second or third c.leavage, no 50 per cent time can be given. Those eggs which completed third cleavage in this concentration of reagent did so with only a moderate delay of about 26 min. The delay in cleavage in 30 per cent of the eggs appears to be a less important effect at 100 pg/ml than the effect of totally blocking development in 70 per cent of the eggs. To learn which stages of early development were sensitive to the inhibitory action of lipoic acid, we exposed the embryos to several concentrations of the reagent, at several early developmental periods. The exposures were made for two-hour periods at O-2, 4-6 and S-10 hr after fertilization, at temperatures between 20” and 23°C. Experimental
Cell Research
38
Nancy
72
Wolfson
and Dorothy
S. Fry
Exposing embryos to 40 ,ug/ml lipoic acid during any of these three developmental periods resulted in only a few abnormal embryos. Exposing embryos to 100 pg/ml lipoic acid at the same three periods resulted in large numbers of abnormal embryos. These abnormalities included a variety of misshapen TABLE III.
Effect
of 2 hr exposures to lipoic acid at three different periods of
early development (Arbacia
punctulata).
A different sample of eggs from a single batch was exposed for each treatment in a given experiment. The treatments consisted of exposing the embryos to 100 ,og/ml lipoic acid in sea water for 2 hr, at one of three different periods of development. At the temperature prevailing, 22223’C, 10 hr after fertilization corresponds to hatching time. Two to three days later the embryos were examined for abnormalities. The percentage of all abnormal embryos resulting from each treatment is shown in the “abn.” column. The percentage of abnormal embryos with clearly defined extensions resulting from each treatment is shown in the “ext.” column. Time O-2
hr
after 4-6
fertilization hr
X-10
hr
Expt.
abn.
ext.
abn.
ext.
abn.
ext.
1 2 3 ‘I 5
22 18 17 30 32
I) 0 0 0 0
76 100 X3 92 36
0 23 9 40 10
24 7 11 8 6
0 0 3 0 0
Ave.
24
0
67
16
11
-rl
and delayed embryos, as well as embryos with clearly defined extensions of the type shown in Fig. 1 b. The percentage of abnormal embryos including those with extensions and the percentage of only those with extensions were counted two to four days after fertilization and recorded in Table III. The number of abnormal embryos resulting from exposure to lipoic acid in the period 4-6 hr after fertilization averages 67 per cent and is significantly greater than abnormalities resulting from earlier and later exposures (24 per cent for O-2 hr and 11 per cent for 8-10 hr). The embryos with extensions occurred only in cultures exposed during the 4-6 hr period, in all experiments save one. The average occurrence of this particular abnormality after the early exposure was 0 per cent, after the middle exposure was 16 per cent, and after the third exposure was < 1 per cent. Both the extended embryos, considered Experimental
Cell Research
38
Lipoic
acid on echinoderm
embryos
alone, and the total abnormalities are highest after 4-6 hr post-fertilization. These results demonstrate to the reagent at this time.
73
the exposure a maximum
period at sensitivity
DISCUSSION
Oxidized lipoic acid affects cleavage very little at concentrations which have a marked effect on subsequent morphogenesis in the echinoderm embryos tested (4 pg/ml). This is in accord with reports on amphibian development which responds to lipoic acid by abnormal morphogenesis, while mitoses remain unaffected [3]. A higher concentration of lipoic acid, 100 pg/ml, did not affect the rate of cleavage of the sand dollar through the third cleavage, although the cleavage of sea urchin eggs suffered delay and disintegration by the second cleavage. The differing sensitivity of the two echinoids to lipoic acid may depend to some extent on the temperatures at which cleavage is optimal for each, 13-15°C for Echinnrachnius and 20-23°C for Arbctcirr, with a resultant more extended development time for the sand dollar. Ham and Eakin [4] report that the regeneration of tentacles of hydras exposed to lipoic acid at room temperature is inhibited, while if exposed at 3”C, regeneration is normal. They interpret the variation of effect with temperature as indicative of interference with active metabolism by lipoic acid. In both sand dollar and sea urchin, 100 pg/ml lipoic acid has very little efl’ect on the first cleavage. This makes it an unlikely participant in the cleavage inhibition associated \vith egg homogenates, since homogenates frequently delay the first cleavage quite appreciably [8]. 11’hen embryos are exposed continuously to low doses of lipoic acid (1 lug/ml for the sand dollar and 40 rug/ml for the sea urchin) development is generally blocked at the blastula stage. \\‘hen the embryos are exposed to the reagent for a shorter period during early development abnormal embryos develop subsequently. Runnstrijm [6] has described in detail the developmental anomalies, including exogastrulae, which result from treatments of lipoic acid on eggs of Parncen trotus liuidns. The developmental period most susceptible to the disturbing influence of lipoic acid occurs before the late blastula stage in the sea urchin and sand dollar. For both these forms, the period of maximum sensitivity is thus \vell before any morphological manifestation of disturbances. In Arbcrcicr the period 1-6 hr after fertilization is more sensitive to lipoic acid than earlier and later pre-blastula periods. This concurs with the period of maximum sensitivity to other reagents which disturb morphogenesis in sea urchin eggs, Experimental
Cell
Research
38
74
Nancy
Wolfson
and Dorothy
S. Fry
in particular, iodosobenzoic acid which produces its maximum effect 3-6 hr after fertilization in Psammechinus miliaris [l 1. Both iodosobenzoic acid and oxidized lipoic acid are sulfhydryl oxidants. Backstrom [2] has suggested that iodosobenzoic acid very probably oxidizes sulfhydryl components on the surface of blastomeres rather than in the interior of the cells. Similarly lipoic acid may also be oxidizing components on the surface of the blastomeres which are required for normal movement of the cells at gastrulation. If the action of the oxidized lipoic acid is limited to the cell surface, then by simultaneous or subsequent treatments with reducing agents it should prove possible to prevent or reverse the effects of lipoic acid on development. SUMMARY
1. Lipoic acid, a naturally occurring cyclic disultide, induces abnormal development, especially at the time of gastrulation in Echinarachnius parma and Arabacia punctulata. 2. No delay in cleavage is produced in E. parma by lipoic acid in concentrations up to 1O-3 M. In A. punctulata a moderate delay is produced in the cleavage of some eggs at concentrations which also block cleavage entirely in many eggs after one or a few initial cleavages. 3. The portion of the developmental period sensitive to the reagent occurs before blastulation while the morphological manifestations of the deleterious action occur at the blastula stage or later in E. parma. 4. In A. punctulata the developmental period most subject to the influence of lipoic acid in producing abnormal embryos occurs after the first 2 hr of development and at least 2 hr before the blastulae hatch. 5. The action of lipoic acid produced during the sensitive period is not reversed by removing the embryos to fresh sea water in either the sea urchin or sand dollar. We wish to thank the staff at the several marine laboratories concerned for the facilities and assistance they provided. The investigation was supported by grant E-301 from American Cancer Society. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
B~CKSTR~~M, S., Arkiu 2002. II 4, 485 (1953). Exptt Cell Res. 16, 165 (1959). BRACHET, J., Develop. Riot. 7, 348 (1963). HAM, R. G. and EAKIN, R. E., J. Exptl Zooi. 139, 55 (1958). HENDERSON, R. F. and EAKIN, R. E., J. Exptl Zoot. 141, 175 (1959). RUNNSTR~~M, J., Exptl Cell Res. 11, 660 (1956). WOLFSON, N., Exptl Cell Res. 18, 504 (1959). WOLFSON, N. and WILBUR, K. M., ibid. 21, 219 (1960).
Experimental
Cell Research
38
Experimental
THE
EFFECT DEVELOPING NANCY
OF
OXIDIZED
LIPOIC
ECHINODERM WOLFSON
and DOROTHY
Department of Physiology, Mt. Department of Biology, Randolph-Macon Received
Cell Research
ACID
38, 66- 74 (1965)
ON
EMBRYOS S. FRY
Holyoke College S. Ha&y, Mass., and Woman’s College Lynchburg, Va., U.S.A. June 9, 1964
IN earlier studies we observed that homogenates of sea urchin eggs contained a cleavage inhibitor whose inhibitory action was intensified in the presence of sulfhydryl reagents [7, 81. In attempting to identify the cleavage inhibitor, we became interested in various naturally occurring sulfhydryl and disulfide compounds, and their effect on mitosis. Lipoic acid in its oxidized form is a cyclic disulfide well known for its role in oxidative decarboxylations. Recently it has been studied in a different context, that of its effect on morphogenetic and regenerative processes [3, 4, 5, 61. To distinguish between possible effects on cell division and on differentiation we have applied lipoic acid to developing echinoderm eggs whose early cleavages can be readily observed and timed. The experiments described indicate that low concentrations of oxidized lipoic acid have only slight effect on early cleavage but produce a marked inhibition of normal derelopment in sand dollar and sea urchin embryos. METHODS The observations were made on eggs of the sand dollar, Echinarachnius parnla, and of the sea urchin, Arbacia punctulata. The sand dollars were collected in Maine, at the Mount Desert Island Biological Laboratory, and the sea urchins in Connecticut and North Carolina, at the Marine Research Laboratory of the University of Connecticut, and the Duke University Marine Laboratory. The observations on Echinarachnius were made in July, and those on Arbacia, for the most part, in early spring. The lipoic acid was supplied in its oxidized form by General Biochemicals, Chagrin Falls, Ohio. A solution of the slightly soluble acid was prepared by converting it to the sodium salt with NaOH and neutralizing with HCl. A stock solution contained 2000 pg lipoic acid per ml in 0.04 M NaOH, 0.04 111 HCl, at pH 8, and was kept refrigerated until discarded after one week. 1 Present Experimental
address:
Department
Cell Research
38
of Zoology,
McGill
University,
Montreal,
Canada.
Lip&
acid on echinoderm
67
embryos
RESULTS
Experiments with Echinarachnius parma.-The eggs of the sand dollar, E. parma, are large and transparent, and the morphological transitions are much easier to follow than in the smaller, pigmented eggs of Arbacia. Shortly after fertilization the eggs were added to sea water containing various concentrations of lipoic acid, from lO-’ to 10~~ M. In these concentrations embryos developed normally up to the hatching blastula stage. The control embryos proceeded through hatching, gastrulation and pluteus formation while the embryos exposed to the lipoic acid solution remained as blastulae, spinning actively within their fertilization membranes. The inhibited embryos remained intact, often spinning for one or two days after their counterparts in sea water had formed long-armed plutei. Total and consistent blocking of this type was obtained at concentrations of 10P5 M. Some embryos were blocked at 1OF M and fewer were blocked at lo-’ M. Embryos exposed from fertilization through blastula stage to 1O-5 M lipoic. acid, and then removed to fresh sea water, remained in their inhibited state. The blocking was irreversible at that concentration for that duration of exposure. We observed little delay in cleavage rate in eggs developing in concentrations up to 200 ,ug lipoic acid (lop3 M). By counting eggs cleaved at several intervals, and by plotting these counts against time, rate curves are obtained and a very accurate time of 50 per cent cleavage found for controls and experimentals. The results of one such experiment to observe the effect of three concentrations of lipoic acid on the rate of early cleavage are shown in Table I. TABLE
I. Effect
lipoic acid on cleavage rate (Echinarachnius
of
parma).
The time at which 50 per cent of the eggs in any dish completed their second and third cleavages is shown in min after fertilization, for controls and experimental embryos exposed to three concentrations of lipoic acid (13°C). The delay accumulated by the experimental embryos at the end of the third cleavage is the difference in min as compared with controls. Concentration of lipoic acid kidml
0 4 40 200
Time of 50 y0 cleavage, Cleavage II Cleavage 160 161 164
213 220 220 216
min III
Delay at III
7 7 3
Experimental
Cell Research
38
68
Nancy
Wolfson
and Dorothy
S. Fry
It is apparent that very little delay was produced during the first three cleavages by any of the three concentrations of lipoic acid. Even concentrations of 200 ,ug/ml produced a delay of no more than 3 min by the end of third cleavage. The measurements on cleavage rates confirmed observations during the course of other experiments in which controls and experimentals did not vary in rate of development until after the blastula stage. Having found that lipoic acid at lo- 5 M had no discernible effect on cleavage rate or form but blocked further development at the spinning blastula stage, we next tried to find whether some stages of early development were especially sensitive to the inhibitory action of the reagent. Eggs were fertilized and exposed to 4 pg/ml of lipoic acid in sea water within 15 min after fertilization. Aliquots of eggs were removed from the lipoic-sea water at intervals, washed and placed in fresh sea water. Eggs removed from lipoicsea water at any time after completion of second cleavage generally showed characteristic abnormalities of development. Eggs removed before second cleavage usually did not show many such abnormalities. The duration of the exposure, from 15 min after fertilization through second cleavage, seemed a rough minimum exposure for obtaining a morphological effect, (about 2 hr at 15’C). We used the 2 hr interval for “pulse” exposures distributed over the developmental period from fertilization to gastrulation. Several abnormalities characterize the later development of embryos that have undergone short-term exposure to lipoic acid. These include (I) a delay in gastrulation, (2) disorganized mesenchymal proliferation into the blastocoel, (3) frequent occurrence of exogastrulae, and (4) abnormalities in the arms of individuals reaching the pluteus stage. We chose the appearance of exogastrulae as an easily recognizable criterion for assessing the effects of lipoic acid on development (Fig. 1 a). Two exogastrulae are shown in the figure. The embryo on the left shows well defined constrictions in the proximal region of the endoderm. The distal regions of the everted tube are less well defined, terminating in loose clusters of cells which tend to adhere to clusters on other exogastrulae, as shown in the photograph. The percentage of exogastrulae from samples of eggs exposed for 2 hr to lipoic acid at various times during development was determined for several egg batches. The results are tabulated in Fig. 2. Although the percentages Fig. l.-Characteristic abnormalities to lipoic acid for 2 hr during early lipoic acid for 2 hr, beginning shortly exposed to 100 ,ug/ml lipoic acid for Microscopic magnification x 200 for Experimental
Cell
Research
38
resulting from exposure of developing echinoderm embryos development. (a) Echinarachnius parma exposed to 4 pg/ml after fertilization. Age: 2 days at 15’C. (b) Arbacia punctulata 2 hr, beginning 4 hr after fertilization. Age: 4 days at 2O’C. both species.
Lipoic acid on echinoderm
embryos
Experimental
Cell
70
Nancy
Wolfson
and Dorothy
S. Fry
are too variable to distinguish among the sensitivities of the first four exposure periods, there is no question of the abrupt drop-off in sensitivity that occurs if the exposure to lipoic acid is made after the eighth hr of development. It is also clear from Fig. 2 that embryos exposed during early cleavages are sensitive to the adverse action of lipoic acid on their subsequent development, even though, as we have shown above, their cleavage rate was unaffected. Finally, we can note that some embryos are affected irreversibly by the lipoic acid, no matter which early developmental stage is exposed. Experiments with Arbacia punctulata.-Eggs of the sea urchin, A. punctulatu, were fertilized and lo-15 min later were transferred to sea water with TABLE
II. Effect
of tipoic acid on cleavage rate (Arbacia
punctulata).
The time at which 50 per cent of the eggs in any dish completed their first, second and third cleavages is shown in min after fertilization, for controls and experimental embryos exposed to three concentrations of lipoic acid (23°C). The delay accumulated by the experimental embryos at the end of the third cleavage is the difference in min as compared with controls. Eggs exposed to 100 pg/ml lipoic acid never achieved 50 per cent cleavage but began cleaving at the approximate time shown in parentheses. Concentration of lipoic acid &ml
Cleavage 67 68 68 70
0
4 40 100
Time
of 50 % cleavage,
min
I
II
III
108 110 118 (began 126)
142 146 158 (began 168)
Delay
at III
4 10 (approx.
26)
one of three concentrations of lipoic acid: 4, 40 or 100 rug/ml. At 4 ,ug/ml the embryos were slightly delayed in their development but eventually tormed normal plutei. At 40 pg/ml the embryos did not form plutei, and most of them stopped developing at, or before the blastula stage. The blocking of development was not as clearcut in Arbacia as in Echinarachnius. Many of the inhibited Arbacia embryos were irregular in form, some were unhatched and others were disintegrating within a few hours after exposure to the lipoic acid solution. At the highest concentration tested, 100 pg/ml, the embryos disintegrated before reaching blastula stage. At this concentration the deleterious effect of the reagent was noted by the time of second cleavage. The inhibition of early cleavage was more apparent in Arbacia than Echinarachnius (Table II). The delay in cleavage resulting from the lower c,oncentrations of lipoic acid was small but with 100 ,ug/ml, many embryos Experimental
Cell
Research
38
Lipoic
acid on echinoderm
71
embryos
had their cleavages blocked from the third cleavage on. For the experiment in Table II, at 100 pg/ml, nearly all the eggs completed first cleavage but less than 30 per cent of the embryos cleaved more than once. The time reported is that when the eggs began to cleave, and since fewer than 30 per Developmental
Hours 0
I
2
3
4
5
6
Periods
Expt. I.
of ter 7
46
fertilization
8
9
IO
of exposure
Percentage I2
stages
51
12
to lipoic
acid
I3
I4
I5
I6
I7
I8
I9
exogastrulae
75
2.
5
3.
0
4.
0
5.
-22
ave.
II
8
21
30
5
51 2
d!.iO~~~
23
37
0
0
0
0
0
0
0
0
0
0
0
0
--
26
0
I
0
Fig. 2.-The stages drawn in the top row of diagrams are: fertilized egg, 4-cell, l&cell, early blastula, mid-blastula, spinning blastula, late blastula, early gastrula. They are placed according to their occurrence in time after fertilization at 15°C. The duration and time of exposure of the embryos to 4 pg/ml lipoic acid are indicated by horizontal bars. A different sample from a single batch of fertilized eggs was exposed for each treatment in a given experiment. The percentage of exogastrulae resulting from each treatment is tabulated under the appropriate exposure period (Echinarachnius parma.)
cent completed second or third c.leavage, no 50 per cent time can be given. Those eggs which completed third cleavage in this concentration of reagent did so with only a moderate delay of about 26 min. The delay in cleavage in 30 per cent of the eggs appears to be a less important effect at 100 pg/ml than the effect of totally blocking development in 70 per cent of the eggs. To learn which stages of early development were sensitive to the inhibitory action of lipoic acid, we exposed the embryos to several concentrations of the reagent, at several early developmental periods. The exposures were made for two-hour periods at O-2, 4-6 and S-10 hr after fertilization, at temperatures between 20” and 23°C. Experimental
Cell Research
38
Nancy
72
Wolfson
and Dorothy
S. Fry
Exposing embryos to 40 ,ug/ml lipoic acid during any of these three developmental periods resulted in only a few abnormal embryos. Exposing embryos to 100 pg/ml lipoic acid at the same three periods resulted in large numbers of abnormal embryos. These abnormalities included a variety of misshapen TABLE III.
Effect
of 2 hr exposures to lipoic acid at three different periods of
early development (Arbacia
punctulata).
A different sample of eggs from a single batch was exposed for each treatment in a given experiment. The treatments consisted of exposing the embryos to 100 ,og/ml lipoic acid in sea water for 2 hr, at one of three different periods of development. At the temperature prevailing, 22223’C, 10 hr after fertilization corresponds to hatching time. Two to three days later the embryos were examined for abnormalities. The percentage of all abnormal embryos resulting from each treatment is shown in the “abn.” column. The percentage of abnormal embryos with clearly defined extensions resulting from each treatment is shown in the “ext.” column. Time O-2
hr
after 4-6
fertilization hr
X-10
hr
Expt.
abn.
ext.
abn.
ext.
abn.
ext.
1 2 3 ‘I 5
22 18 17 30 32
I) 0 0 0 0
76 100 X3 92 36
0 23 9 40 10
24 7 11 8 6
0 0 3 0 0
Ave.
24
0
67
16
11
-rl
and delayed embryos, as well as embryos with clearly defined extensions of the type shown in Fig. 1 b. The percentage of abnormal embryos including those with extensions and the percentage of only those with extensions were counted two to four days after fertilization and recorded in Table III. The number of abnormal embryos resulting from exposure to lipoic acid in the period 4-6 hr after fertilization averages 67 per cent and is significantly greater than abnormalities resulting from earlier and later exposures (24 per cent for O-2 hr and 11 per cent for 8-10 hr). The embryos with extensions occurred only in cultures exposed during the 4-6 hr period, in all experiments save one. The average occurrence of this particular abnormality after the early exposure was 0 per cent, after the middle exposure was 16 per cent, and after the third exposure was < 1 per cent. Both the extended embryos, considered Experimental
Cell Research
38
Lipoic
acid on echinoderm
embryos
alone, and the total abnormalities are highest after 4-6 hr post-fertilization. These results demonstrate to the reagent at this time.
73
the exposure a maximum
period at sensitivity
DISCUSSION
Oxidized lipoic acid affects cleavage very little at concentrations which have a marked effect on subsequent morphogenesis in the echinoderm embryos tested (4 pg/ml). This is in accord with reports on amphibian development which responds to lipoic acid by abnormal morphogenesis, while mitoses remain unaffected [3]. A higher concentration of lipoic acid, 100 pg/ml, did not affect the rate of cleavage of the sand dollar through the third cleavage, although the cleavage of sea urchin eggs suffered delay and disintegration by the second cleavage. The differing sensitivity of the two echinoids to lipoic acid may depend to some extent on the temperatures at which cleavage is optimal for each, 13-15°C for Echinnrachnius and 20-23°C for Arbctcirr, with a resultant more extended development time for the sand dollar. Ham and Eakin [4] report that the regeneration of tentacles of hydras exposed to lipoic acid at room temperature is inhibited, while if exposed at 3”C, regeneration is normal. They interpret the variation of effect with temperature as indicative of interference with active metabolism by lipoic acid. In both sand dollar and sea urchin, 100 pg/ml lipoic acid has very little efl’ect on the first cleavage. This makes it an unlikely participant in the cleavage inhibition associated \vith egg homogenates, since homogenates frequently delay the first cleavage quite appreciably [8]. 11’hen embryos are exposed continuously to low doses of lipoic acid (1 lug/ml for the sand dollar and 40 rug/ml for the sea urchin) development is generally blocked at the blastula stage. \\‘hen the embryos are exposed to the reagent for a shorter period during early development abnormal embryos develop subsequently. Runnstrijm [6] has described in detail the developmental anomalies, including exogastrulae, which result from treatments of lipoic acid on eggs of Parncen trotus liuidns. The developmental period most susceptible to the disturbing influence of lipoic acid occurs before the late blastula stage in the sea urchin and sand dollar. For both these forms, the period of maximum sensitivity is thus \vell before any morphological manifestation of disturbances. In Arbcrcicr the period 1-6 hr after fertilization is more sensitive to lipoic acid than earlier and later pre-blastula periods. This concurs with the period of maximum sensitivity to other reagents which disturb morphogenesis in sea urchin eggs, Experimental
Cell
Research
38
74
Nancy
Wolfson
and Dorothy
S. Fry
in particular, iodosobenzoic acid which produces its maximum effect 3-6 hr after fertilization in Psammechinus miliaris [l 1. Both iodosobenzoic acid and oxidized lipoic acid are sulfhydryl oxidants. Backstrom [2] has suggested that iodosobenzoic acid very probably oxidizes sulfhydryl components on the surface of blastomeres rather than in the interior of the cells. Similarly lipoic acid may also be oxidizing components on the surface of the blastomeres which are required for normal movement of the cells at gastrulation. If the action of the oxidized lipoic acid is limited to the cell surface, then by simultaneous or subsequent treatments with reducing agents it should prove possible to prevent or reverse the effects of lipoic acid on development. SUMMARY
1. Lipoic acid, a naturally occurring cyclic disultide, induces abnormal development, especially at the time of gastrulation in Echinarachnius parma and Arabacia punctulata. 2. No delay in cleavage is produced in E. parma by lipoic acid in concentrations up to 1O-3 M. In A. punctulata a moderate delay is produced in the cleavage of some eggs at concentrations which also block cleavage entirely in many eggs after one or a few initial cleavages. 3. The portion of the developmental period sensitive to the reagent occurs before blastulation while the morphological manifestations of the deleterious action occur at the blastula stage or later in E. parma. 4. In A. punctulata the developmental period most subject to the influence of lipoic acid in producing abnormal embryos occurs after the first 2 hr of development and at least 2 hr before the blastulae hatch. 5. The action of lipoic acid produced during the sensitive period is not reversed by removing the embryos to fresh sea water in either the sea urchin or sand dollar. We wish to thank the staff at the several marine laboratories concerned for the facilities and assistance they provided. The investigation was supported by grant E-301 from American Cancer Society. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
B~CKSTR~~M, S., Arkiu 2002. II 4, 485 (1953). Exptt Cell Res. 16, 165 (1959). BRACHET, J., Develop. Riot. 7, 348 (1963). HAM, R. G. and EAKIN, R. E., J. Exptl Zooi. 139, 55 (1958). HENDERSON, R. F. and EAKIN, R. E., J. Exptl Zoot. 141, 175 (1959). RUNNSTR~~M, J., Exptl Cell Res. 11, 660 (1956). WOLFSON, N., Exptl Cell Res. 18, 504 (1959). WOLFSON, N. and WILBUR, K. M., ibid. 21, 219 (1960).
Experimental
Cell Research
38