- Email: [email protected]
Precocious induction of activation responses in amphibian oocytes by divalent ionophore A23187
DEVELOPMENTAL
Precocious
BIOLOGY
45, 378-381
Induction
(1975)
of Activation Divalent
Responses
lonophore
University,
Oocytes
by
A23 187
ANN M. BELANGERANDALLEN W. The Johns Hopkins
in Amphibian
School of Hygiene and Public Health, Maryland 21205
SCHUETZ 615 North
Wolfe Street, Baltimore,
Accepted April 7,1975 Temporal relationships between maturational events and the onset of activation in response to divalent ionophore and to pricking were examined following in vitro exposure of Rana pipiens oocytes to desoxycorticosterone acetate (DOCA). Activation was evaluated on the basis of vitelline envelope elevation and cortical granule breakdown. Ionophore-induced activation was first observed after 18 hr of DOCA incubation, coincident with the time of separation of the vitelline envelope from the oocyte surface and 2-3 hr after breakdown of the germinal vesicle. Activation in response to pricking was not observed until 30 hr of DOCA incubation. Neither ionophore treatment nor pricking resulted in activation of oocytes that had not been incubated with DOCA. These results indicate that oocytes can be activated many hours earlier than previously demonstrated. The time of onset of the capacity for activation appears to be related to germinal vesicle breakdown and vitelline envelope separation. INTRODUCTION
mones (3, 7, 10). Under these conditions, the interval between germinal vesicle breakdown and activation in response to pricking is approximately 15 hr in Rana pipiens oocytes (1). To further elucidate relationships between activation and maturation, we have compared the timing of ionophore-induced activation with that of activation by pricking in relation to events of hormone-induced meiotic and physiological maturation.
Amphibian oocyte activation typically occurs following fertilization or artificial stimulation, such as pricking with a glass needle. Distinguishing characteristics of the activation response include breakdown of cortical granules and elevation of the vitelline envelope. Recently, treatment with divalent ionophore A23187 has also been shown to induce activation of oocytes of Xenopus laevis and a variety of other species (11, 12). Under natural conditions, fertilization takes place after oocytes have undergone nuclear maturation (germinal vesicle breakdown) and have reached second metaphase of meiosis. Activation in response to pricking apparently begins to occur at the same meiotic stage (13). Although this suggests nuclear control of the capacity for activation, enucleated oocytes can also be activated (9). Thus, the relative importance of cytoplasmic and nuclear events in regulating the timing of activation remains to be determined. These problems can be readily investigated in amphibians, since oocytes can be induced to undergo meiotic maturation in vitro by treatment with particular steroid hor-
MATERIALS
METHODS
Ovaries were removed from large, hibernating Rana pipiens and placed in amphibian Ringer’s salt solution. Subsequent manipulations and incubations were carried out in this medium. Fully grown oocytes were dissected from individual follicles so that the vitelline envelope could be visualized. Oocytes collected from an individual animal were pooled and randomly distributed into petri dishes. Experiments were performed on oocytes from three animals. Desoxycorticosterone acetate (DOCA) was added to oocytes at a concentration of 1 fig/ml to induce nuclear breakdown and reinitiate the meiotic process. 378
Copyright 0 1975by Academic Press, Inc. All rights of reproduction in any form reserved
AND
379
BRIEFNOTES
At regular intervals after addition of hormone, groups of ten oocytes from each animal were transferred to amphibian Ringer’s and either treated with ionophore A23187 [dissolved initially in dimethyl sulfoxide (DMSO) and added to a concentration of 1 wg/ml amphibian Ringer’s], treated with DMSO alone, pricked with a glass needle or left untreated. In addition, ten oocytes per animal which had not been exposed to hormone were treated with ionophore after 22 hr of incubation in amphibian Ringers. Activation, as evidenced by vitelline envelope elevation, was scored after 15 min of treatment. Both treated and control oocytes were then fixed in 10% formalin and dissected to determine whether the nucleus had broken down and/or the vitelline envelope had separated from the oocyte surface. (The vitelline envelope separates from the oocyte plasma membrane close to the time of nuclear breakdown (8). Separation is not to be confused with vitelline envelope elevation, which occurs upon activation and is characterized by marked expansion of the perivitelline space.) At certain times, additional treated and control oocytes were
fixed in 4% glutaraldehyde, postfixed in 1% osmium tetroxide and embedded in a mixture of Epon and Araldite. One-twomicron sections were prepared, stained with Alcian blue and examined for the presence of cortical granules. RESULTS
Activation (vitelline envelope elevation) in response to ionophore treatment was first observed after 18 hr of DOCA incubation. In contrast, activation by pricking was not observed until 30 hr of incubation in DOCA (Fig. 1). Onset of ionophoreinduced activation appeared to coincide with the time of vitelline envelope separation, which occurred 2-3 hr after germinal vesicle breakdown (Fig. 2). Induction of activation by ionophore was never observed in control oocytes (not treated with DOCA) or in maturing oocytes which had not yet undergone nuclear breakdown and vitelline envelope separation. Likewise, none of the oocytes treated with DMSO alone was activated. Ionophore treatment has been shown to induce cortical contraction in Rana pipiens oocytes divested of vitelline envelopes (6).
100
r
Time
FIG. 1. Timing of activation
of
lncubotlon
in DOCA
(Hrs)
in response to ionophore and to pricking following in vitro incubation of oocytes with DOCA (1 pglml). Oocytes were treated with ionophore A23187 (1 fig/ml) or pricked with a glass needle at each designated time. After 15 min, the percentage of oocytes exhibiting viteiline envelope elevation was determined. A total of 30 oocytes (lo/animal) was examined at each time.
380
DEVELOPMENTALBIOLOGY
VOLUME 45, 1975
100 r
i”i,+dL-d
12
14
16
l
I
!
I
1
I
I
(
1
I
18
20
22
24
26
28
30
32
34
36
Time
of lncubotlon
in DOCA (tirs)
FIG. 2. Timing of germinal vesicle breakdown and vitelline envelope separation following in vitro incubation of oocytes with DOCA (1 pg/ml). Oocytes were fixed in 10% formalin at designated times and were subsequently dissected to determine whether the germinal vesicle was present and/or the vitelline envelope was separated from the oocyte plasma membrane. A total of 30 oocytes (lo/animal) was examined at each time.
In the present experiments, cortical contraction was also observed but only in those oocytes that were activated by ionophore treatment. Histological examination revealed that oocytes treated with ionophore after nuclear maturation (22 hr of DOCA) had undergone cortical granule breakdown as well as vitelline envelope elevation. However, those which were treated with ionophore prior to nuclear maturation (14 hr of DOCA) did not exhibit cortical granule breakdown or vitelline envelope elevation. Pricking of oocytes at 36 hr of incubation in DOCA resulted in both vitelline envelope elevation and cortical granule breakdown; neither response was observed following pricking at 22 hr. Thus, the activation response to ionophore as well as to pricking, includes both vitelline envelope elevation and cortical granule breakdown.
oocytes can be activated by pricking. Presumably, ionophore-induced activation involves release, uptake or intracellular redistribution of divalent cations (5, 11). Although the specific cation responsible for activation induction has not been identified, several studies indicate that changes in the distribution of calcium ions are important for fertilization and artificial activation (11, 12, 14). Hormone-induced changes which make oocytes responsive to ionophore could include increases in the availability of divalent cations or in the sensitivity of the cortical granules and/or oocyte surface to these ions. Cytoplasmic factors, which form in response to hormone and appear to mediate the maturation process (4), might also regulate the timing of activation. The close temporal association between vitelline envelope separation and ionophore-induced activation suggests that detachment of the vitelline envelope DISCUSSION makes the oocyte responsive to ionophore. Present experiments demonstrate that Alternatively, the onset of sensitivity to ionophore A23187 begins to activate oo- ionophore could be a direct, although cytes as soon as they have undergone somewhat delayed, result of nuclear breaknuclear breakdown and vitelline envelope down. However, this seems unlikely since separation, which is nearly 14 hr before the ability of oocytes to activate, at least in
BRIEF NOTES
381
response to pricking, is not impaired by possibility that these maturation processes involve changes in divalent ions reenucleation (9). Regardless of the nature of the events mains to be investigated. regulating the time of ionophore-induced This investigation was supported by grants from activation, it is clear that they are com- the Population Council (No. 4732.2093.10) and the plete nearly 14 hr before those necessary for Ford Foundation and by a training grant from the activation by pricking. This may be a National Institutes of Health (No. 5TOl HD00109-09). consequence of differences in the mech- We thank R. L. Hamill of Eli Lilly Co., Indianapolis, ar+ms by which ionophore and pricking IN, for providing ionophore A23187 used in this study. induce activation. In this regard, it has REFERENCES recently been observed that sea urchin 1. BELANGER, A. M., and SCHUETZ, A. W. (1974). J. oocytes exhibit a cortical reaction only in Cell Biol. 63, 19a. the portion of the oocyte surface in direct 2. CHAMBERS, E. L., PRESSMAN,B. C., and ROSE, B. (1974). J. Cell Biol. 63, 56a. contact with ionophore (2). Apparently, ionophore affects individual cortical gran- 3. MASUJ Y. (1967). J. Exp. Zoo!. 166, 365-376. 4. MASUI, Y., and MARKERT, C. L. (1971). J. Exp. ules without inducing the wave of cortical Zool. 177, 129-145. granule breakdown characteristic of fertili5. REED, P. W., and LARDY, H. A. (1972). In “The zation or activation by pricking. Thus, the Role of Membranes in Metabolic Regulation” (M. A. Mehlman and R. W. Hanson, eds.), pp. substantial interval between the onset of 111-131. Academic Press, New York. sensitivity to ionophore and to pricking 6. SCHROEDER, T. E., and STRICKLAND, D. L. (1974). may include maturation processes related Erp. Cell Res. 83, 139-142. to the ability of the oocyte surface to 7. SCHUETZ, A. W. (1967). J. Exp. Zool. 166,347-354, translate a pricking stimulus into a wave of 8. SCHUETZ,A. W. (1972). Biol. Reprod. 6,67-77. 9. SMITH, L. D., and ECKER, R. E. (1969). Deuelop. cortical granule breakdown. Biol. 19, 281-309. In conclusion, these experiments demon10. SMITH, L. D., ECKER, R. E., and SUBT~LNY, S. strate that the times at which oocytes can (1968). Deuelop. Biol. 17, 627-643. undergo activation in response to pricking 11. STEINHARDT, R. A., and EPEL, D. (1974). Proc. or ionophore are fixed in relation to a Nat. Acad. Sci. USA 71, 1915-1919. program of differentiation initiated by ste- 12. STEINHARDT, R. A., EPEL, D., CARROLL, E. J., JR., and YANACIMACHI, R. (1974). Nature (London) roid hormone. Furthermore, activation in 252,41-43. response to pricking appears to require 13. SUBTELNY, S., and BRADT, C. (1961). Deuelop. maturation processes not necessary for Biol. 3, 96-114. activation in response to ionophore. The 14. WOLF, D. P. (1974). Deuelop. Biol. 40, 102-115.
Precocious
BIOLOGY
45, 378-381
Induction
(1975)
of Activation Divalent
Responses
lonophore
University,
Oocytes
by
A23 187
ANN M. BELANGERANDALLEN W. The Johns Hopkins
in Amphibian
School of Hygiene and Public Health, Maryland 21205
SCHUETZ 615 North
Wolfe Street, Baltimore,
Accepted April 7,1975 Temporal relationships between maturational events and the onset of activation in response to divalent ionophore and to pricking were examined following in vitro exposure of Rana pipiens oocytes to desoxycorticosterone acetate (DOCA). Activation was evaluated on the basis of vitelline envelope elevation and cortical granule breakdown. Ionophore-induced activation was first observed after 18 hr of DOCA incubation, coincident with the time of separation of the vitelline envelope from the oocyte surface and 2-3 hr after breakdown of the germinal vesicle. Activation in response to pricking was not observed until 30 hr of DOCA incubation. Neither ionophore treatment nor pricking resulted in activation of oocytes that had not been incubated with DOCA. These results indicate that oocytes can be activated many hours earlier than previously demonstrated. The time of onset of the capacity for activation appears to be related to germinal vesicle breakdown and vitelline envelope separation. INTRODUCTION
mones (3, 7, 10). Under these conditions, the interval between germinal vesicle breakdown and activation in response to pricking is approximately 15 hr in Rana pipiens oocytes (1). To further elucidate relationships between activation and maturation, we have compared the timing of ionophore-induced activation with that of activation by pricking in relation to events of hormone-induced meiotic and physiological maturation.
Amphibian oocyte activation typically occurs following fertilization or artificial stimulation, such as pricking with a glass needle. Distinguishing characteristics of the activation response include breakdown of cortical granules and elevation of the vitelline envelope. Recently, treatment with divalent ionophore A23187 has also been shown to induce activation of oocytes of Xenopus laevis and a variety of other species (11, 12). Under natural conditions, fertilization takes place after oocytes have undergone nuclear maturation (germinal vesicle breakdown) and have reached second metaphase of meiosis. Activation in response to pricking apparently begins to occur at the same meiotic stage (13). Although this suggests nuclear control of the capacity for activation, enucleated oocytes can also be activated (9). Thus, the relative importance of cytoplasmic and nuclear events in regulating the timing of activation remains to be determined. These problems can be readily investigated in amphibians, since oocytes can be induced to undergo meiotic maturation in vitro by treatment with particular steroid hor-
MATERIALS
METHODS
Ovaries were removed from large, hibernating Rana pipiens and placed in amphibian Ringer’s salt solution. Subsequent manipulations and incubations were carried out in this medium. Fully grown oocytes were dissected from individual follicles so that the vitelline envelope could be visualized. Oocytes collected from an individual animal were pooled and randomly distributed into petri dishes. Experiments were performed on oocytes from three animals. Desoxycorticosterone acetate (DOCA) was added to oocytes at a concentration of 1 fig/ml to induce nuclear breakdown and reinitiate the meiotic process. 378
Copyright 0 1975by Academic Press, Inc. All rights of reproduction in any form reserved
AND
379
BRIEFNOTES
At regular intervals after addition of hormone, groups of ten oocytes from each animal were transferred to amphibian Ringer’s and either treated with ionophore A23187 [dissolved initially in dimethyl sulfoxide (DMSO) and added to a concentration of 1 wg/ml amphibian Ringer’s], treated with DMSO alone, pricked with a glass needle or left untreated. In addition, ten oocytes per animal which had not been exposed to hormone were treated with ionophore after 22 hr of incubation in amphibian Ringers. Activation, as evidenced by vitelline envelope elevation, was scored after 15 min of treatment. Both treated and control oocytes were then fixed in 10% formalin and dissected to determine whether the nucleus had broken down and/or the vitelline envelope had separated from the oocyte surface. (The vitelline envelope separates from the oocyte plasma membrane close to the time of nuclear breakdown (8). Separation is not to be confused with vitelline envelope elevation, which occurs upon activation and is characterized by marked expansion of the perivitelline space.) At certain times, additional treated and control oocytes were
fixed in 4% glutaraldehyde, postfixed in 1% osmium tetroxide and embedded in a mixture of Epon and Araldite. One-twomicron sections were prepared, stained with Alcian blue and examined for the presence of cortical granules. RESULTS
Activation (vitelline envelope elevation) in response to ionophore treatment was first observed after 18 hr of DOCA incubation. In contrast, activation by pricking was not observed until 30 hr of incubation in DOCA (Fig. 1). Onset of ionophoreinduced activation appeared to coincide with the time of vitelline envelope separation, which occurred 2-3 hr after germinal vesicle breakdown (Fig. 2). Induction of activation by ionophore was never observed in control oocytes (not treated with DOCA) or in maturing oocytes which had not yet undergone nuclear breakdown and vitelline envelope separation. Likewise, none of the oocytes treated with DMSO alone was activated. Ionophore treatment has been shown to induce cortical contraction in Rana pipiens oocytes divested of vitelline envelopes (6).
100
r
Time
FIG. 1. Timing of activation
of
lncubotlon
in DOCA
(Hrs)
in response to ionophore and to pricking following in vitro incubation of oocytes with DOCA (1 pglml). Oocytes were treated with ionophore A23187 (1 fig/ml) or pricked with a glass needle at each designated time. After 15 min, the percentage of oocytes exhibiting viteiline envelope elevation was determined. A total of 30 oocytes (lo/animal) was examined at each time.
380
DEVELOPMENTALBIOLOGY
VOLUME 45, 1975
100 r
i”i,+dL-d
12
14
16
l
I
!
I
1
I
I
(
1
I
18
20
22
24
26
28
30
32
34
36
Time
of lncubotlon
in DOCA (tirs)
FIG. 2. Timing of germinal vesicle breakdown and vitelline envelope separation following in vitro incubation of oocytes with DOCA (1 pg/ml). Oocytes were fixed in 10% formalin at designated times and were subsequently dissected to determine whether the germinal vesicle was present and/or the vitelline envelope was separated from the oocyte plasma membrane. A total of 30 oocytes (lo/animal) was examined at each time.
In the present experiments, cortical contraction was also observed but only in those oocytes that were activated by ionophore treatment. Histological examination revealed that oocytes treated with ionophore after nuclear maturation (22 hr of DOCA) had undergone cortical granule breakdown as well as vitelline envelope elevation. However, those which were treated with ionophore prior to nuclear maturation (14 hr of DOCA) did not exhibit cortical granule breakdown or vitelline envelope elevation. Pricking of oocytes at 36 hr of incubation in DOCA resulted in both vitelline envelope elevation and cortical granule breakdown; neither response was observed following pricking at 22 hr. Thus, the activation response to ionophore as well as to pricking, includes both vitelline envelope elevation and cortical granule breakdown.
oocytes can be activated by pricking. Presumably, ionophore-induced activation involves release, uptake or intracellular redistribution of divalent cations (5, 11). Although the specific cation responsible for activation induction has not been identified, several studies indicate that changes in the distribution of calcium ions are important for fertilization and artificial activation (11, 12, 14). Hormone-induced changes which make oocytes responsive to ionophore could include increases in the availability of divalent cations or in the sensitivity of the cortical granules and/or oocyte surface to these ions. Cytoplasmic factors, which form in response to hormone and appear to mediate the maturation process (4), might also regulate the timing of activation. The close temporal association between vitelline envelope separation and ionophore-induced activation suggests that detachment of the vitelline envelope DISCUSSION makes the oocyte responsive to ionophore. Present experiments demonstrate that Alternatively, the onset of sensitivity to ionophore A23187 begins to activate oo- ionophore could be a direct, although cytes as soon as they have undergone somewhat delayed, result of nuclear breaknuclear breakdown and vitelline envelope down. However, this seems unlikely since separation, which is nearly 14 hr before the ability of oocytes to activate, at least in
BRIEF NOTES
381
response to pricking, is not impaired by possibility that these maturation processes involve changes in divalent ions reenucleation (9). Regardless of the nature of the events mains to be investigated. regulating the time of ionophore-induced This investigation was supported by grants from activation, it is clear that they are com- the Population Council (No. 4732.2093.10) and the plete nearly 14 hr before those necessary for Ford Foundation and by a training grant from the activation by pricking. This may be a National Institutes of Health (No. 5TOl HD00109-09). consequence of differences in the mech- We thank R. L. Hamill of Eli Lilly Co., Indianapolis, ar+ms by which ionophore and pricking IN, for providing ionophore A23187 used in this study. induce activation. In this regard, it has REFERENCES recently been observed that sea urchin 1. BELANGER, A. M., and SCHUETZ, A. W. (1974). J. oocytes exhibit a cortical reaction only in Cell Biol. 63, 19a. the portion of the oocyte surface in direct 2. CHAMBERS, E. L., PRESSMAN,B. C., and ROSE, B. (1974). J. Cell Biol. 63, 56a. contact with ionophore (2). Apparently, ionophore affects individual cortical gran- 3. MASUJ Y. (1967). J. Exp. Zoo!. 166, 365-376. 4. MASUI, Y., and MARKERT, C. L. (1971). J. Exp. ules without inducing the wave of cortical Zool. 177, 129-145. granule breakdown characteristic of fertili5. REED, P. W., and LARDY, H. A. (1972). In “The zation or activation by pricking. Thus, the Role of Membranes in Metabolic Regulation” (M. A. Mehlman and R. W. Hanson, eds.), pp. substantial interval between the onset of 111-131. Academic Press, New York. sensitivity to ionophore and to pricking 6. SCHROEDER, T. E., and STRICKLAND, D. L. (1974). may include maturation processes related Erp. Cell Res. 83, 139-142. to the ability of the oocyte surface to 7. SCHUETZ, A. W. (1967). J. Exp. Zool. 166,347-354, translate a pricking stimulus into a wave of 8. SCHUETZ,A. W. (1972). Biol. Reprod. 6,67-77. 9. SMITH, L. D., and ECKER, R. E. (1969). Deuelop. cortical granule breakdown. Biol. 19, 281-309. In conclusion, these experiments demon10. SMITH, L. D., ECKER, R. E., and SUBT~LNY, S. strate that the times at which oocytes can (1968). Deuelop. Biol. 17, 627-643. undergo activation in response to pricking 11. STEINHARDT, R. A., and EPEL, D. (1974). Proc. or ionophore are fixed in relation to a Nat. Acad. Sci. USA 71, 1915-1919. program of differentiation initiated by ste- 12. STEINHARDT, R. A., EPEL, D., CARROLL, E. J., JR., and YANACIMACHI, R. (1974). Nature (London) roid hormone. Furthermore, activation in 252,41-43. response to pricking appears to require 13. SUBTELNY, S., and BRADT, C. (1961). Deuelop. maturation processes not necessary for Biol. 3, 96-114. activation in response to ionophore. The 14. WOLF, D. P. (1974). Deuelop. Biol. 40, 102-115.