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The role of the maturation-promoting factor in controlling protein synthesis in Xenopus oocytes
DEVELOPMENTAL
BIOLOGY
90,
445-447
(1982)
The Role of the Maturation-Promoting Factor in Controlling Synthesis in Xenopus Oocytes WILLIAM Department Received
of Biology, November
University 9, 1981;
Protein
J. WASSERMAN of Rochester,
accepted
in revised
Rochester,
fwm
New
York
January
14627
11, 1982
Oocyte cytosol, containing maturation-promoting factor activity, induces a twofold increase in the rate of protein synthesis as well as inducing germinal vesicle breakdown (GVBD) when microinjected into Xenopus oocytes. In the current study, it is shown that the cytosol activity responsible for inducing the increase in protein synthesis can be separated from the activity that induces GVBD in recipient oocytes.
INTRODUCTION
dialysis against 0.025 M ,&glycerophosphate, pH 6.5, 0.035 M NaF, 0.15 M NaCl, kO.002 M EGTA, 0.0025 M MgS04, and 0.001 M ATP, the fractionated cytosol was stored in aliquots at -70°C. Cytosol fractions were injected into oocytes with a multiple injection micropipet (Wasserman and Masui, 1975). Protein synthesis measurements. Control, progesterone-treated, MPF- and cytoplasm-injected recipient oocytes were injected with [3H]leucine (sp act, 4 Ci/mmole, Amersham) at 0.2 &i/oocyte. The absolute rate of protein synthesis was determined as previously described (Wasserman et al., 1982).
Xenopus laevis oocytes treated with progesterone undergo a twofold increase in the absolute rate of protein synthesis approximately 1 to 2 hr prior to germinal vesicle breakdown (GVBD) (Wasserman et al., 1982). It has been demonstrated that this increase is due primarily to the recruitment of additional mRNA onto polysomes (Richter et al., 1982). Furthermore, it has been shown that the microinjection of cytosol, containing maturation-promoting factor (MPF), can induce an equivalent increase in protein synthesis in recipient oocytes (Wasserman e2:al., 1982). These cytosol preparations also induce GVBD when injected into oocytes (Wasserman and Masui, 1976; Drury, 1978; Wu and Gerhart, 1980). In the current study, an attempt has been made to determine if the activity in MPF preparations which induces GVBD can be sleparated from the activity which stimulates protein synthesis. MATERIALS
AND
RESULTS
AND
DISCUSSION
In a previous study, it was demonstrated that cytosols, prepared from mature Xenopus oocytes, could induce a twofold increase in the protein synthetic rate in recipient oocytes (Wasserman et al., 1982). To further test this phenomenon, experiments were conducted to compare the effect that whole cytoplasm and cytosol injections have on recipient oocyte protein synthesis. As seen in Table 1, control stage VI oocytes had a protein synthetic rate of 21.1 ng hr-l per oocyte, while the rate in oocytes treated with progesterone (postGVBD) was 1.7-fold greater (36.2 ng hr-’ per oocyte). These values are essentially the same as those reported earlier (Wasserman et al., 1982). Oocytes injected with 40 nl of cytoplasm, taken from control oocytes, did not undergo GVBD and had a protein synthetic rate that was equivalent to noninjected control oocytes (Table 1). Therefore, the injection process itself has no effect on protein synthesis. On the other hand, the injection of cytoplasm, taken from mature oocytes (post-GVBD), produced a 1.8-fold increase in the recipient oocyte protein synthetic rate and all oocytes underwent GVBD
METHODS
Ovarian
material. MLanually defolliculated stage VI oocytes (Dumont, 1972) were cultured in OR2 medium (Wallace et al., 1973). Oocytes that were stimulated with progesterone were placed in OR-2 containing the steroid at 1 pg/ml for continuous exposure. Preparation of MPF cytosols. Cytosols containing MPF activity were prepared from in vitro matured Xenopus oocytes with an extraction buffer which contained 0.025 M P-glycerophosphate, pH 6.5, 0.20 M NaF, 0.004 M EGTA, 0.001 M Mg SO1, and 0.25 M sucrose as previously described (Wasserman et al., 1982). In certain cases, cytosols were prepared in the absence of EGTA. Ammonium sulfate fractionation was accomplished by adding solid (NH&SO, to the crude cytosol. Following
Xenopus
445 0012-1606/82/040445-03$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
446
DEVELOPMENTAL BIOLOGY
TABLE 1 PROTEIN SYNTHESIS AND GVBD IN RESPONSE TO CYTOPLASMIC AND CYTOSOL INJECTIONS Injected material (40 nl) None, control None, progesterone Control cytoplasm Mature cytoplasm Control cytosol Mature cytosol Dialysis buffer
Recipient oocyte protein synthesis (ng hr-‘/oocyte) 21.1 36.2 19.7 35.3 18.0 40.9 25.1
f 2.5 -t 6.6 f 2.9 + 4.4 (1) f 5.2 (1)
(3) (3)" (3) (3)b (3)*
Recipient oocyte GVBD O/48 48/48 O/48 48/48 O/16 48148 O/16
aProtein synthesis was measured 7 hr after progesterone was added, the time of GVBD. Mean + SD. Number of determinations in parentheses. ‘Protein synthesis was measured following GVBD, 3 hr following the injection of cytoplasm or cytosol.
(Table 1). This synthetic rate is identical to the rate in progesterone-matured oocytes (Table 1). The 150,OOOg cytosol (20- to 40% ammonium sulfate fraction) made from control stage VI oocytes did not have an effect on protein synthesis nor on GVBD when injected into oocytes (18 ng hr-’ per oocyte). On the contrary, cytosol (20-to-40% cut) made from in. vitro matured oocytes (post-GVBD) had a pronounced stimulatory effect on the protein synthetic rate and on GVBD (Table 1). Therefore, the extraction process itself does not create or activate this “protein synthesis stimulatory factor” (PSSF). PSSF can only be detected in cytosol made from mature oocytes. However, the protein synthetic rate in oocytes receiving active cytosol was always slightly higher than the rate in progesterone-matured sibling oocytes and in sibling oocytes that were injected with mature cytoplasm (Table 1). This may be due to the concentrating of PSSF in the cytosol preparations. The active cytosol preparations used so far, induce both the increase in protein synthesis and GVBD in recipient oocytes. One would like to know if the same substance or substances in the cytosol are inducing both events or whether these two physiological changes can be attributed to separate entities in the injected preparation. Several procedures are known to inactivate the cytosol as far as its ability to induce GVBD in oocytes. These include, the addition of calcium ions, repeated freezing and thawing, and prolonged storage of the cytosol at 4°C (Wasserman and Masui, 1976). It is not known what effect these treatments have on PSSF activity. Cytosol was prepared and dialyzed against buffer lacking EGTA (see Materials and Methods). CaCl, (100 PM) was added to one aliquot and left at 2°C for 1 hr.
VOLUME 90, 1982
Another aliquot was frozen and thawed three times, while a third aliquot was left in the cold room at 4°C for 10 days. All three aliquots were assayed for their ability to induce GVBD and stimulate protein synthesis in recipient oocytes. As seen in Table 2, cytosol prepared in the absence of EGTA still had a pronounced stimulatory effect on protein synthesis and GVBD. However, cytosol treated with calcium ions, or subjected to repeated freezing and thawing, or prolonged storage at 4°C showed a greatly diminished capacity to induce GVBD (Table 2). On the contrary, the ability of these cytosols to stimulate protein synthesis was barely affected. All three recipient oocyte protein synthetic rates were still at least 1.6 times higher than the control rate (Tables 1 and 2). Thus, it appears that the effect the cytosol has on protein synthesis and on GVBD can be separated from each other at a functional level. However, the same substance(s) in the cytosol may have the ability to catalyze both reactions, but one process may be more susceptible to inactivation. Therefore, an attempt was made to physically separate these two activities assuming that they are indeed associated with two separate substances or group of substances in the cytosol. The cytosol preparations used so far have been subjected to ammonium sulfate fractionation. It was previously determined that the majority of MPF activity, based on the ability to induce GVBD, comes out of solution between 20 and 40% ammonium sulfate saturation at 2°C. The possibility existed that the two activities in question could be separated from each other with a more refined fractionation scheme. Cytosol prepared with EGTA was subjected to ammonium sulfate precipitation producing fractions at 20-40, 20-30, and 30-40% saturation. Each fraction was dissolved in different volumes of dialysis buffer to give approximately equivalent protein concentrations. Following dialysis, each fraction was assayed for its effect on protein synthesis and on GVBD in recipient oocytes. As seen in Table 3, PSSF activity and GVBD activity can be found TABLE 2 INACTIVATION OF GVBD ACIWITY BUT NOT PSSF ACTIVITY IN OOCYTE
Mature oocyte cytosol treatmenta None 100 & Ca2+ Freeze-thaw (3~) 4°C (10 days)
CYTOSOLS
Recipient oocyte protein synthesis (ng hr-‘/oocyte) 38.8 32.9 34.1 35.3
+ 5.8 zk 9.2 f 7.7 + 4.6
(3) (3) (3) (3)
Recipient oocyte GVBD 48148 2/48 5/48 O/48
“All cytosols were prepared in the absence of EGTA, 20-to-40% ammonium sulfate fraction.
447
BRIEF NOTES
AMMONIUM
TABLE 3 SULFATE FRACTIONATION AND GVBD ACTIVITIES
Ammonium sulfate fraction (% saturation)
Recipient oocyte pr’otein synthesis (ng hr-‘/oocyte)
Whole 20-40 40-60 20-30 30-40
44.3 40.9 24.3 27.8 35.6
cytosol”
a 150,OOOg supernatant, cytosol *Values come from Table 1.
k 2 + rt +
6.2 5.2 2.3 2.7 4.3
OF PSSF
Recipient oocyte GVBD
(3) (3)* (3) (3) (3)
made
48/48 48/48 4/48 40148 6/48 with
when injected with cytoplasm containing MPF (Wasserman and Masui, 1975; Drury and Schorderet-Slatkine, 1975). Therefore, it appears that the component inducing GVBD can still function in a cell without the increase in protein synthesis. Thus, it is now possible to isolate two factors from mature oocytes. One appears to play a role in controlling protein synthesis during oocyte maturation involving mRNA recruitment (Richter et al., 1982). The second appears to play a direct or almost direct role in causing the dissolution of the nuclear envelope.
EGTA.
in the 20-to-40% ammonium sulfate fraction. However, it appears that the majority of the activity responsible for inducing GVBD can be segregated to the 20-to-30% fraction, while the majority of PSSF activity can be found in the 30-to40% fraction. One must note that there is some overlap of the two activities, but it appears that these two activities can be separated from each other at the physical level. Therefore, the ability to stimulate protein synthesis must reside with a substance or group of substances that are physically distinct from those responsible for inducing GVBD in oocytes. The separation of these two activities, at least at the functional level, has already been inferred in the literature. First, stage VI oocytes, taken from an animal that has recently ovulated (4 days), have an elevated rate of protein synthesis (33 ng hr-’ per oocyte) yet these oocytes do not undergo GVBD or ovulation. These “stimulated” oocytes still have to be treated with progesterone in vitro in order to induce a precocious GVBD (Wasserman, et al., 1982). Therefore, the twofold increase in protein synthesis is not sufficient to trigger the dissolution of the nuclear envelope. Secondly, it has been shown that oocytes, whose protein synthesis was inhibited by cycloheximide, could still undergo GVBD
This Society
work was supported by grants from the American Cancer (CD-112) and the National Science Foundation (PCM-8104491).
REFERENCES DRURY, K. C. (1978). Method for the preparation of active maturation promotional factor (MPF) from in witro maturing oocytes of Xenopus laevis. Differentiation 10, 181-186. DRURY, K. C., and SCHORDERET-SLATKINE, S. (1975). Effects of cycloheximide on the autocatalytic nature of the maturation promotional factor (MPF) from in vitro maturing oocytes of Xenqpus Levis. Cell 4, 268-274. DUMONT, J. N. (1972). Oogenesis in Xenqwus .!.a&.~ (Daudin). 1. Stage of oocyte development in laboratory maintained animals. J. Marphol. 136, 153-180. RICHTER, J. D., WASSERMAN, W. J., and SMITH, L. D. (1982). The mechanism for increased protein synthesis during Xenqm-s oocyte maturation. Deuelup. Biol. 89, 159-167. WALLACE, R. A., JARED, D. W., DUMONT, J. N., and SEGA, M. W. (1973). Protein incorporation by isolated amphibian occytes. III. Optimum incubation conditions. J. Exp. Zool. 184, 321-334. WASSERMAN, W. J., and MASU~, Y. (1975). Effects of cycloheximide on a cytoplasmic factor initiating meiotic maturation in Xenopus oocytes. Exp. Cell Res. 91, 381-388. WASSERMAN, W. J., and MASUI, Y. (1976). A cytoplasmic factor promoting oocyte maturation: Its extraction and preliminary characterization. Science 191, 1266-1268. WASSERMAN, W. J., RICHTER, J. D., and SMITH, L. D. (1982). Protein synthesis during maturation promoting factorand progesteroneinduced maturation in Xenspus oocytes. Develop. Biol 89, 152-158. Wu, M., and GERHART, J. (1980). Partial purification and characterization of the maturation-promoting factor from eggs of Xenopus Levis. Develop. Biol. 79, 465-477.
BIOLOGY
90,
445-447
(1982)
The Role of the Maturation-Promoting Factor in Controlling Synthesis in Xenopus Oocytes WILLIAM Department Received
of Biology, November
University 9, 1981;
Protein
J. WASSERMAN of Rochester,
accepted
in revised
Rochester,
fwm
New
York
January
14627
11, 1982
Oocyte cytosol, containing maturation-promoting factor activity, induces a twofold increase in the rate of protein synthesis as well as inducing germinal vesicle breakdown (GVBD) when microinjected into Xenopus oocytes. In the current study, it is shown that the cytosol activity responsible for inducing the increase in protein synthesis can be separated from the activity that induces GVBD in recipient oocytes.
INTRODUCTION
dialysis against 0.025 M ,&glycerophosphate, pH 6.5, 0.035 M NaF, 0.15 M NaCl, kO.002 M EGTA, 0.0025 M MgS04, and 0.001 M ATP, the fractionated cytosol was stored in aliquots at -70°C. Cytosol fractions were injected into oocytes with a multiple injection micropipet (Wasserman and Masui, 1975). Protein synthesis measurements. Control, progesterone-treated, MPF- and cytoplasm-injected recipient oocytes were injected with [3H]leucine (sp act, 4 Ci/mmole, Amersham) at 0.2 &i/oocyte. The absolute rate of protein synthesis was determined as previously described (Wasserman et al., 1982).
Xenopus laevis oocytes treated with progesterone undergo a twofold increase in the absolute rate of protein synthesis approximately 1 to 2 hr prior to germinal vesicle breakdown (GVBD) (Wasserman et al., 1982). It has been demonstrated that this increase is due primarily to the recruitment of additional mRNA onto polysomes (Richter et al., 1982). Furthermore, it has been shown that the microinjection of cytosol, containing maturation-promoting factor (MPF), can induce an equivalent increase in protein synthesis in recipient oocytes (Wasserman e2:al., 1982). These cytosol preparations also induce GVBD when injected into oocytes (Wasserman and Masui, 1976; Drury, 1978; Wu and Gerhart, 1980). In the current study, an attempt has been made to determine if the activity in MPF preparations which induces GVBD can be sleparated from the activity which stimulates protein synthesis. MATERIALS
AND
RESULTS
AND
DISCUSSION
In a previous study, it was demonstrated that cytosols, prepared from mature Xenopus oocytes, could induce a twofold increase in the protein synthetic rate in recipient oocytes (Wasserman et al., 1982). To further test this phenomenon, experiments were conducted to compare the effect that whole cytoplasm and cytosol injections have on recipient oocyte protein synthesis. As seen in Table 1, control stage VI oocytes had a protein synthetic rate of 21.1 ng hr-l per oocyte, while the rate in oocytes treated with progesterone (postGVBD) was 1.7-fold greater (36.2 ng hr-’ per oocyte). These values are essentially the same as those reported earlier (Wasserman et al., 1982). Oocytes injected with 40 nl of cytoplasm, taken from control oocytes, did not undergo GVBD and had a protein synthetic rate that was equivalent to noninjected control oocytes (Table 1). Therefore, the injection process itself has no effect on protein synthesis. On the other hand, the injection of cytoplasm, taken from mature oocytes (post-GVBD), produced a 1.8-fold increase in the recipient oocyte protein synthetic rate and all oocytes underwent GVBD
METHODS
Ovarian
material. MLanually defolliculated stage VI oocytes (Dumont, 1972) were cultured in OR2 medium (Wallace et al., 1973). Oocytes that were stimulated with progesterone were placed in OR-2 containing the steroid at 1 pg/ml for continuous exposure. Preparation of MPF cytosols. Cytosols containing MPF activity were prepared from in vitro matured Xenopus oocytes with an extraction buffer which contained 0.025 M P-glycerophosphate, pH 6.5, 0.20 M NaF, 0.004 M EGTA, 0.001 M Mg SO1, and 0.25 M sucrose as previously described (Wasserman et al., 1982). In certain cases, cytosols were prepared in the absence of EGTA. Ammonium sulfate fractionation was accomplished by adding solid (NH&SO, to the crude cytosol. Following
Xenopus
445 0012-1606/82/040445-03$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
446
DEVELOPMENTAL BIOLOGY
TABLE 1 PROTEIN SYNTHESIS AND GVBD IN RESPONSE TO CYTOPLASMIC AND CYTOSOL INJECTIONS Injected material (40 nl) None, control None, progesterone Control cytoplasm Mature cytoplasm Control cytosol Mature cytosol Dialysis buffer
Recipient oocyte protein synthesis (ng hr-‘/oocyte) 21.1 36.2 19.7 35.3 18.0 40.9 25.1
f 2.5 -t 6.6 f 2.9 + 4.4 (1) f 5.2 (1)
(3) (3)" (3) (3)b (3)*
Recipient oocyte GVBD O/48 48/48 O/48 48/48 O/16 48148 O/16
aProtein synthesis was measured 7 hr after progesterone was added, the time of GVBD. Mean + SD. Number of determinations in parentheses. ‘Protein synthesis was measured following GVBD, 3 hr following the injection of cytoplasm or cytosol.
(Table 1). This synthetic rate is identical to the rate in progesterone-matured oocytes (Table 1). The 150,OOOg cytosol (20- to 40% ammonium sulfate fraction) made from control stage VI oocytes did not have an effect on protein synthesis nor on GVBD when injected into oocytes (18 ng hr-’ per oocyte). On the contrary, cytosol (20-to-40% cut) made from in. vitro matured oocytes (post-GVBD) had a pronounced stimulatory effect on the protein synthetic rate and on GVBD (Table 1). Therefore, the extraction process itself does not create or activate this “protein synthesis stimulatory factor” (PSSF). PSSF can only be detected in cytosol made from mature oocytes. However, the protein synthetic rate in oocytes receiving active cytosol was always slightly higher than the rate in progesterone-matured sibling oocytes and in sibling oocytes that were injected with mature cytoplasm (Table 1). This may be due to the concentrating of PSSF in the cytosol preparations. The active cytosol preparations used so far, induce both the increase in protein synthesis and GVBD in recipient oocytes. One would like to know if the same substance or substances in the cytosol are inducing both events or whether these two physiological changes can be attributed to separate entities in the injected preparation. Several procedures are known to inactivate the cytosol as far as its ability to induce GVBD in oocytes. These include, the addition of calcium ions, repeated freezing and thawing, and prolonged storage of the cytosol at 4°C (Wasserman and Masui, 1976). It is not known what effect these treatments have on PSSF activity. Cytosol was prepared and dialyzed against buffer lacking EGTA (see Materials and Methods). CaCl, (100 PM) was added to one aliquot and left at 2°C for 1 hr.
VOLUME 90, 1982
Another aliquot was frozen and thawed three times, while a third aliquot was left in the cold room at 4°C for 10 days. All three aliquots were assayed for their ability to induce GVBD and stimulate protein synthesis in recipient oocytes. As seen in Table 2, cytosol prepared in the absence of EGTA still had a pronounced stimulatory effect on protein synthesis and GVBD. However, cytosol treated with calcium ions, or subjected to repeated freezing and thawing, or prolonged storage at 4°C showed a greatly diminished capacity to induce GVBD (Table 2). On the contrary, the ability of these cytosols to stimulate protein synthesis was barely affected. All three recipient oocyte protein synthetic rates were still at least 1.6 times higher than the control rate (Tables 1 and 2). Thus, it appears that the effect the cytosol has on protein synthesis and on GVBD can be separated from each other at a functional level. However, the same substance(s) in the cytosol may have the ability to catalyze both reactions, but one process may be more susceptible to inactivation. Therefore, an attempt was made to physically separate these two activities assuming that they are indeed associated with two separate substances or group of substances in the cytosol. The cytosol preparations used so far have been subjected to ammonium sulfate fractionation. It was previously determined that the majority of MPF activity, based on the ability to induce GVBD, comes out of solution between 20 and 40% ammonium sulfate saturation at 2°C. The possibility existed that the two activities in question could be separated from each other with a more refined fractionation scheme. Cytosol prepared with EGTA was subjected to ammonium sulfate precipitation producing fractions at 20-40, 20-30, and 30-40% saturation. Each fraction was dissolved in different volumes of dialysis buffer to give approximately equivalent protein concentrations. Following dialysis, each fraction was assayed for its effect on protein synthesis and on GVBD in recipient oocytes. As seen in Table 3, PSSF activity and GVBD activity can be found TABLE 2 INACTIVATION OF GVBD ACIWITY BUT NOT PSSF ACTIVITY IN OOCYTE
Mature oocyte cytosol treatmenta None 100 & Ca2+ Freeze-thaw (3~) 4°C (10 days)
CYTOSOLS
Recipient oocyte protein synthesis (ng hr-‘/oocyte) 38.8 32.9 34.1 35.3
+ 5.8 zk 9.2 f 7.7 + 4.6
(3) (3) (3) (3)
Recipient oocyte GVBD 48148 2/48 5/48 O/48
“All cytosols were prepared in the absence of EGTA, 20-to-40% ammonium sulfate fraction.
447
BRIEF NOTES
AMMONIUM
TABLE 3 SULFATE FRACTIONATION AND GVBD ACTIVITIES
Ammonium sulfate fraction (% saturation)
Recipient oocyte pr’otein synthesis (ng hr-‘/oocyte)
Whole 20-40 40-60 20-30 30-40
44.3 40.9 24.3 27.8 35.6
cytosol”
a 150,OOOg supernatant, cytosol *Values come from Table 1.
k 2 + rt +
6.2 5.2 2.3 2.7 4.3
OF PSSF
Recipient oocyte GVBD
(3) (3)* (3) (3) (3)
made
48/48 48/48 4/48 40148 6/48 with
when injected with cytoplasm containing MPF (Wasserman and Masui, 1975; Drury and Schorderet-Slatkine, 1975). Therefore, it appears that the component inducing GVBD can still function in a cell without the increase in protein synthesis. Thus, it is now possible to isolate two factors from mature oocytes. One appears to play a role in controlling protein synthesis during oocyte maturation involving mRNA recruitment (Richter et al., 1982). The second appears to play a direct or almost direct role in causing the dissolution of the nuclear envelope.
EGTA.
in the 20-to-40% ammonium sulfate fraction. However, it appears that the majority of the activity responsible for inducing GVBD can be segregated to the 20-to-30% fraction, while the majority of PSSF activity can be found in the 30-to40% fraction. One must note that there is some overlap of the two activities, but it appears that these two activities can be separated from each other at the physical level. Therefore, the ability to stimulate protein synthesis must reside with a substance or group of substances that are physically distinct from those responsible for inducing GVBD in oocytes. The separation of these two activities, at least at the functional level, has already been inferred in the literature. First, stage VI oocytes, taken from an animal that has recently ovulated (4 days), have an elevated rate of protein synthesis (33 ng hr-’ per oocyte) yet these oocytes do not undergo GVBD or ovulation. These “stimulated” oocytes still have to be treated with progesterone in vitro in order to induce a precocious GVBD (Wasserman, et al., 1982). Therefore, the twofold increase in protein synthesis is not sufficient to trigger the dissolution of the nuclear envelope. Secondly, it has been shown that oocytes, whose protein synthesis was inhibited by cycloheximide, could still undergo GVBD
This Society
work was supported by grants from the American Cancer (CD-112) and the National Science Foundation (PCM-8104491).
REFERENCES DRURY, K. C. (1978). Method for the preparation of active maturation promotional factor (MPF) from in witro maturing oocytes of Xenopus laevis. Differentiation 10, 181-186. DRURY, K. C., and SCHORDERET-SLATKINE, S. (1975). Effects of cycloheximide on the autocatalytic nature of the maturation promotional factor (MPF) from in vitro maturing oocytes of Xenqpus Levis. Cell 4, 268-274. DUMONT, J. N. (1972). Oogenesis in Xenqwus .!.a&.~ (Daudin). 1. Stage of oocyte development in laboratory maintained animals. J. Marphol. 136, 153-180. RICHTER, J. D., WASSERMAN, W. J., and SMITH, L. D. (1982). The mechanism for increased protein synthesis during Xenqm-s oocyte maturation. Deuelup. Biol. 89, 159-167. WALLACE, R. A., JARED, D. W., DUMONT, J. N., and SEGA, M. W. (1973). Protein incorporation by isolated amphibian occytes. III. Optimum incubation conditions. J. Exp. Zool. 184, 321-334. WASSERMAN, W. J., and MASU~, Y. (1975). Effects of cycloheximide on a cytoplasmic factor initiating meiotic maturation in Xenopus oocytes. Exp. Cell Res. 91, 381-388. WASSERMAN, W. J., and MASUI, Y. (1976). A cytoplasmic factor promoting oocyte maturation: Its extraction and preliminary characterization. Science 191, 1266-1268. WASSERMAN, W. J., RICHTER, J. D., and SMITH, L. D. (1982). Protein synthesis during maturation promoting factorand progesteroneinduced maturation in Xenspus oocytes. Develop. Biol 89, 152-158. Wu, M., and GERHART, J. (1980). Partial purification and characterization of the maturation-promoting factor from eggs of Xenopus Levis. Develop. Biol. 79, 465-477.