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The use of Xenopus oocytes for the bioassay of ras
METHODS: A Companion to Methods in Enzymology Vol. 1, No. 3, December, pp. 315-318, 1990
The Use of Xenopus Oocytes for the Bioassay of ras Michael Wu,* Rosalind Kim, ~ and Sung-Hou K i m ~'* Departments of *Molecular and Cell Biology and *Chemistry, University of California, Berkeley, California 94720; and tLawrence Berkeley Laboratory, Berkeley, California 94720
Fully grown Xenopus laevis oocytes dissected from ovaries are at a stationary state of meiotic prophase arrest. They can be triggered to enter meiosis by various agents such as progesterone, which acts through the outer membrane, or oncogenic ras protein, which acts upon injection. This process, the meiotic maturation of the oocyte that results in the breakdown of the nucleus or the germinal vesicle, is the essence of the biological assay for ras activity. © 1990AcademicPress,Inc.
The South African clawed frog, Xenopus laevis, has large oocytes (1.1-1.3 mm in diameter) which are easily manipulated, injected, and observed under a dissecting microscope. In the natural course of events, gonadotrophic hormone from the pituitary body induces progesterone secretion by the follicle cells surrounding the oocyte. This releases the full-grown (stage VI) oocyte from meiotic prophase arrest (G2 block), and the egg enters meiotic maturation (1, 2). When this is completed, the oocyte is at the second meiotic metaphase and then becomes a fertilizable egg upon ovulation. The meiotic maturation of Xenopus oocytes can be induced in vitro by many agents other than progesterone (3). Injecting oncogenic ras into immature oocytes induces the maturation characteristics that are similar to those induced by progesterone (4). Six to twelve hours after the ras injection, the enlarged nucleus, termed the germinal vesicle (GV), starts to migrate toward the animal pole, displacing the dark pigmentation, and a circular white spot appears. Soon after, a dark ring surrounding the white spot may form. An hour or two later, a tiny polar body at the center of the white spot can also be observed. At this point, the germinal vesicle is broken down, and condensed chromosomes align with the spindle (4).
METHOD Sources and Maintenance of Xenopus laevis Sexually mature Xenopus females can be purchased from Xenopus I (716 Northside, Ann Arbor, MI 48105) 1046-2023/90 $3.00 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
or Nasco (901 Jamesville Avenue, Fort Atkinson, WI 53538). Frogs are kept underwater between 18 and 20°C and a 12-h dark-light cycle is maintained. Because frogs prefer shady areas, direct sunlight should be avoided. Feeding consists of Purina trout chow (pellet size No. 5) or chopped chicken liver given once or twice a week. The water is changed before and after feedings. Clean, dechlorinated tap water is used to submerge frogs at a depth of 1 foot. One to two frogs are maintained in 1 gallon of water. The quality of the oocytes is correlated directly to the degree of stress in the environment in which the frogs are maintained. Poor-quality oocytes can be flushed out by injecting 800-1000 units of human chorionic gonadotropin to the dorsal lymph sac of the frog to induce ovulation. This allows fresh, small oocytes to increase in size to stage VI in 3 months. Frogs that have just ovulated should not be used for 6-8 weeks to prevent oocytes from undergoing spontaneous maturation during the assay (3).
Obtaining Oocytes A female frog is anesthetized by immersion in 0.1% benzocaine (ethyl-p-aminobenzoate, Sigma) in tap water for 10 min. A 7% benzocaine stock is made with ethanol. This anesthetizes the frog for about 3 h, allowing enough time to do surgery. A slit 1.5 cm long is cut along the lateral rear side (beneath the lateral line organ) of the frog. An incision is made through the loose skin layer and then through the muscle layers. A sterile cotton swab is used to search for ovarian tissue, a multilobe structure enclosing the oocytes. The necessary amount of ovarian tissue is gently pulled out with tweezers and snipped off with curved scissors. The rest of the unused tissue is put back; the muscle layer is sewn first and then the loose skin layer by using silk surgical suture (Ethicon K572 silk cardiovascular suture with a needle size 3-0). The recovering frog is placed in shallow water with her nostrils exposed. After the frog awakens, water is added to submerge the frog. The frog is aIlowed to recover for a month in a clean isolated environment before she is reused again, since the shock of surgery sometimes causes spontaneous ovulation or sensitizes the oocytes to undergo spontaneous 315
316
WU, KIM, AND KIM
maturation. Frogs may be recycled for use many times until depletion of ovarian tissue. Lobes of ovarian tissue are separated, cut open with scissors, stored in modified Ringer's saline solution (100 mM NaC1, 1.8 mM KC1, 2 mM CaC12, 1 mM MgC12, 5 mM NaHC03, pH 7.8) with 50 #g/ml gentamicin (Sigma) at a temperature of 15-20°C, and used within 10 h. Single oocytes are dissected from the ovarian tissue with watchmaker's forceps (Dumont No. 5, Roboz Surgical Instrument Co., Inc.) under a dissecting microscope at 15× magnification. The dissection is to free the oocyte from the thecal and surface epithelium layers while the monolayer of follicle cells remains on the oocyte surface. To achieve this, one forcep holds the stalk where the oocyte attaches to the ovarian tissue while the other forcep acts to tear the layers nearby the stalk in order to unwrap the oocyte (like peeling an orange). Damaged oocytes are discarded because they become sensitized for maturation. Oocytes with only one or two outer layers can be injected, but with difficulty. Oocytes obtained from collagenased ovarian tissue tend to swell, making it difficult for the oocytes to take up the injection volume. Thus, mechanically dissected oocytes have the advantage of being easier to inject and heal better after injection. Oocytes obtained during the spring months are more responsive and more synchronous in reaching maturation. During the winter season, oocytes may take 8-10 h to mature in response to progesterone treatment, while in the spring they may take 3-4 h. Similar results were observed in the assay of oncogenic ras. To obtain more responsive oocytes during the winter months, pregnant mare serum gonadotropin (PMSG, CalBiochem) may be used to sensitize the oocytes. Injection of 25 units of PMSG into the dorsal lymph sac of frogs 24 h before use will generate oocytes that mature in 2.5-3 h in response to progesterone treatment (3). If the above maturation time is shorter than 2.5 h, the risk of spontaneous maturation of untreated oocytes will be very high. Careful monitoring of the progesterone control is very important.
Injection The equipment necessary for microinjection of ras into immature oocytes is listed below: 1. Dissecting microscope, e.g., Wild M8 Zoom stereomicroscope 2. Illuminator, e.g., Dolan-Jenner, Model 190 fiber optic illuminator 3. Micromanipulator, e.g., Narishigi mm-33 4. Micromanipulator stand 5. Needle holder 6. Microburet--syringe type 7. Hamilton syringe, Luer tip, volume, 100 #1 8. Polyethylene tubing PZ-60 (Clay Adams)
9. 60% glycerol for filling syringe 10. Injection needle made from 30-#1 microcap capillary (Drummond Scientific Co., Broomall, PA 19008) 11. Custom-made steel spring for retracting the Hamilton syringe plunger Our instruments and techniques for oocyte microinjection are adopted from Gurdon (5). The preparation and calibration of the microinjection needle are shown in Fig. 1.
Description of oocyte microinjection. Oocytes are placed in a petri dish (or in a petri dish containing agar wells fitted to the size of an oocyte) under a dissecting stereomicroscope. A glass needle loaded with ras approaches the oocyte from the side, at an angle of 45 ° (Fig. 2). Forceps are placed on the opposite side to keep the oocyte immobile when the agar wells are not being used. The micromanipulator advances the needle to pierce the oocyte through the equatorial region and the needle tip should stop at the center of the sphere. Positive pressure is applied through the syringe, which is connected to the needle by polyethylene tubing. Fluid meniscus movement through the needle is monitored (a 1-mm displacement delivers 50 nl of sample). Negative pressure is applied to stop fluid movement through the retraction of the plunger by steel spring action. The needle is retracted from the oocyte and the injection is completed. This system is very reliable and is able to handle very small sample volumes. Commonly, 50 nl of sample is injected into each oocyte. There are more sophisticated instruments designed for oocyte injection, such as one recently available from Drummond Scientific Co. Preparation of ras for oocyte injection. The synthetic human e-H-ras gene is expressed in Escherichia coli and the recombinant protein purified (6). The protein is dialyzed extensively against buffer A (50 mM H-glycerol phosphate, 5 mM MgC12, pH 7.4). The sample is centrifuged for 1 min at 15,000g in an Eppendorf microcentrifuge to remove any particulate matter that could block fluid uptake into the injection needle. Observation and Timing of Germinal Vesicle Breakdown Modified Ringer's saline solution at 21°C provides an optimum condition for maturation after the oocyte receives a ras protein injection. The migration of the nucleus (GV) toward the surface of the animal pole and the dissolution of the nuclear membrane (referred to as germinal vesicle breakdown, GVBD) result in the appearance of a whitish spot at the pigmented animal pole (Fig. 2). To confirm the breakdown of the nuclear membrane, oocytes in question may be fixed with 5% trichloroaeetic acid for 5 min and split open for verification (7). Oncogenic ras at a dosage of 14 ng induces GVBD 612 h after injection, while this process using normal ras can take as long as 40 h for completion and requires a
Xenopus laevis OOCYTES AND ras PROTEINS (a)
317 Animal pole
Mlcroplpet
O_
0
Hand pull
lmm
Hand pull
Heat
Equatorial region GVBD
()
()
A ~ 0,3
F I G . 2. Schematic drawing of a Xenopus oocyte undergoing germinal vesicle breakdown (GVBD) upon microinjection of oncogenic ras. The micropipet containing the protein sample is inserted into the equatorial region of a Xenopus oocyte. GVBD is shown to occur after 8-10 h.
mm
(b)
T o quantitate the activity of ras, the sample must be serially diluted in buffer A. Each dilution is injected into 5-10 oocytes. T h e lowest dilution of injected ras t h a t elicits GVBD is indicative of the strength of the ras sample. A control in which 25 nl of buffer A is injected into oocytes
Heati coil
0.3 mrn -- _ -- 140mm TABLE
O.02mm
1
R e a c t i v a t i o n of H u m a n c-H-ras va112 P r o t e i n i n Mevalonate-Deprived Xenopus Oocytes Rubber stopper as weight
Expt
Actual calibration marks are made w i t h w a t e r insoluble ink and l rnm apart
£
60% Glycerol
Air
Sample is pushed out and moved a few calibration marks
Measure size of drop
F I G . 1. Construction and calibration of capillaries for microinjection. (a) The capillary pipet is heated in the center and hand-pulled. (b) Using a heating coil, the pipet tip is formed by applying a weight on one end during heating. Approximate dimensions of the different sections of the pipet are described. (c) Calibration of capillaries for fluid microinjection.
100-fold increase in dosage. In comparison, progesterone (2 #g/ml)-induced GVBD takes place 1-2 h earlier t h a n t h a t induced by oncogenic ras (4). Because partial response to GVBD is not well defined, GVBD in an oocyte is scored as an all or nothing event.
c-H-ras wu2 (ng)
Mevalonate (ng)
Oocytes with GVBD
1
0 0 0 0
56 28 14 7
0 0 0 0
6/6 6/6 6/6 0/6
2
12.5 1.25 0.125
0 0 0
0 0 0
0/6 O/6 O/6
3
12.5 1.25 0.125
56 56 56
0 0 0
0/6 0/6 0/6
4
0.125 0.125 0.125 0 0.125
28 28 28 28 0
4 40 400 0 0
0
0
4
0/3
0 0
0 0
40 400
0/3 0/3
(c) Polyethylentubing
Compactin (ng)
6/6* 6/6* 6/6" 3/3 0/3
Note. Oocyte injections were performed as described in the text. In all experiments a total volume of 50 nl was injected. Initiation of meiosis was measured visually by the appearance of germinal vesicle breakdown (GVBD), which occurred at 8 h after injection except in experiments labeled with an asterisk, in which GVBD occurred at 5 h after injection. In Experiments 3 and 4, oocytes were injected with compactin (25 nl) and t h e n incubated for 1 h at room temperature to achieve mevalonate deprivation and allow depletion of the isoprene pool; they were t h e n injected with h u m a n c-H-ras vau2 protein (25 nl) or a mixture of this protein and mevalonate (25 nl). Compactin was diluted from a stock solution (25 mg/ml) in 25% ethanol. For each experiment three to five oocytes were activated by progesterone treatment as a control for the ability of the oocytes from a particular frog to initiate meiosis (8).
318
WU, KIM, AND KIM
will rule out spontaneous GVBD induction. Table 1 lists results from experiments involving an oncogenic ras with a valine-for-glycine substitution of position 12 (c-HrasVan2); the drug compactin, which inhibits mevalonate synthesis; and mevalonate (8). GVBD induced by c-Hras w112 can be inhibited by compactin and this inhibition can be overcome by mevalonate, an intermediate of cholesterol biosynthesis. These results support the idea that a farnesyl moiety or its derivative is involved in the modification of the ras protein. Double injections can be given, but the total volume is kept at 50 nl or below.
DISCUSSION I
I
The ability of oncogenic ras to induce oocytes to enter meiosis, resulting in the breakdown of the nucleus, provides a fast and easy way to quantitate the activity of the protein, thus providing a convenient system for screening inhibitors or activators of ras protein under an in vivo condition before testing on an animal system. Oncogenic ras can also be injected into tissue culture cells to induce transformation to provide a qualitative study of its activity (9). The mechanism of ras-induced meiosis in oocytes is
not well understood. An attempt to compare it to progesterone induction showed that ras uses an alternate pathway to trigger meiosis in X e n o p u s oocytes that bypasses changes in intracellular cAMP levels (4). Further understanding of the induction of oocyte maturation by ras might help elucidate how this oncogenic protein functions in tumorigenesis.
REFERENCES I
I
I
1. Masui, Y. (1967) J. Exp. Zool. 166, 365-376. 2. Smith, L. D., Eeker, R. E., and Subtelney, S. (1968) Dev. Biol. 17, 627-643. 3. Smith, L. D. (1989) Development 107, 685-699. 4. Birchmeier, C., Broek, D., and Wiglet, M. (1985) Cell 43,615-621. 5. Gurdon, J. B. (1977) Methods Cell Biol. 16, 125-139.
6. Miura, K., Inoue, Y., Nakamori, H., Iwai, S., Ohtsuka, E., Ikehara, M., Noguchi, S., and Nishimura, S. (1986) Jpn. J. Cancer Res. 77, 45-51. 7. Trahey, M., and McCormick, F. (1987) Science 238, 542-545. 8. Schafer, W. R., Kim, R., Sterne, R., Thorner, J., Kim, S.-H., and Rine, J. (1989) Science 245, 379-385. 9. Feramisco, J. R., Kamata, T., Gross, M., Rosenberg, M., and Sweet, R. W. (1984) Cell 38, 107-117.
The Use of Xenopus Oocytes for the Bioassay of ras Michael Wu,* Rosalind Kim, ~ and Sung-Hou K i m ~'* Departments of *Molecular and Cell Biology and *Chemistry, University of California, Berkeley, California 94720; and tLawrence Berkeley Laboratory, Berkeley, California 94720
Fully grown Xenopus laevis oocytes dissected from ovaries are at a stationary state of meiotic prophase arrest. They can be triggered to enter meiosis by various agents such as progesterone, which acts through the outer membrane, or oncogenic ras protein, which acts upon injection. This process, the meiotic maturation of the oocyte that results in the breakdown of the nucleus or the germinal vesicle, is the essence of the biological assay for ras activity. © 1990AcademicPress,Inc.
The South African clawed frog, Xenopus laevis, has large oocytes (1.1-1.3 mm in diameter) which are easily manipulated, injected, and observed under a dissecting microscope. In the natural course of events, gonadotrophic hormone from the pituitary body induces progesterone secretion by the follicle cells surrounding the oocyte. This releases the full-grown (stage VI) oocyte from meiotic prophase arrest (G2 block), and the egg enters meiotic maturation (1, 2). When this is completed, the oocyte is at the second meiotic metaphase and then becomes a fertilizable egg upon ovulation. The meiotic maturation of Xenopus oocytes can be induced in vitro by many agents other than progesterone (3). Injecting oncogenic ras into immature oocytes induces the maturation characteristics that are similar to those induced by progesterone (4). Six to twelve hours after the ras injection, the enlarged nucleus, termed the germinal vesicle (GV), starts to migrate toward the animal pole, displacing the dark pigmentation, and a circular white spot appears. Soon after, a dark ring surrounding the white spot may form. An hour or two later, a tiny polar body at the center of the white spot can also be observed. At this point, the germinal vesicle is broken down, and condensed chromosomes align with the spindle (4).
METHOD Sources and Maintenance of Xenopus laevis Sexually mature Xenopus females can be purchased from Xenopus I (716 Northside, Ann Arbor, MI 48105) 1046-2023/90 $3.00 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
or Nasco (901 Jamesville Avenue, Fort Atkinson, WI 53538). Frogs are kept underwater between 18 and 20°C and a 12-h dark-light cycle is maintained. Because frogs prefer shady areas, direct sunlight should be avoided. Feeding consists of Purina trout chow (pellet size No. 5) or chopped chicken liver given once or twice a week. The water is changed before and after feedings. Clean, dechlorinated tap water is used to submerge frogs at a depth of 1 foot. One to two frogs are maintained in 1 gallon of water. The quality of the oocytes is correlated directly to the degree of stress in the environment in which the frogs are maintained. Poor-quality oocytes can be flushed out by injecting 800-1000 units of human chorionic gonadotropin to the dorsal lymph sac of the frog to induce ovulation. This allows fresh, small oocytes to increase in size to stage VI in 3 months. Frogs that have just ovulated should not be used for 6-8 weeks to prevent oocytes from undergoing spontaneous maturation during the assay (3).
Obtaining Oocytes A female frog is anesthetized by immersion in 0.1% benzocaine (ethyl-p-aminobenzoate, Sigma) in tap water for 10 min. A 7% benzocaine stock is made with ethanol. This anesthetizes the frog for about 3 h, allowing enough time to do surgery. A slit 1.5 cm long is cut along the lateral rear side (beneath the lateral line organ) of the frog. An incision is made through the loose skin layer and then through the muscle layers. A sterile cotton swab is used to search for ovarian tissue, a multilobe structure enclosing the oocytes. The necessary amount of ovarian tissue is gently pulled out with tweezers and snipped off with curved scissors. The rest of the unused tissue is put back; the muscle layer is sewn first and then the loose skin layer by using silk surgical suture (Ethicon K572 silk cardiovascular suture with a needle size 3-0). The recovering frog is placed in shallow water with her nostrils exposed. After the frog awakens, water is added to submerge the frog. The frog is aIlowed to recover for a month in a clean isolated environment before she is reused again, since the shock of surgery sometimes causes spontaneous ovulation or sensitizes the oocytes to undergo spontaneous 315
316
WU, KIM, AND KIM
maturation. Frogs may be recycled for use many times until depletion of ovarian tissue. Lobes of ovarian tissue are separated, cut open with scissors, stored in modified Ringer's saline solution (100 mM NaC1, 1.8 mM KC1, 2 mM CaC12, 1 mM MgC12, 5 mM NaHC03, pH 7.8) with 50 #g/ml gentamicin (Sigma) at a temperature of 15-20°C, and used within 10 h. Single oocytes are dissected from the ovarian tissue with watchmaker's forceps (Dumont No. 5, Roboz Surgical Instrument Co., Inc.) under a dissecting microscope at 15× magnification. The dissection is to free the oocyte from the thecal and surface epithelium layers while the monolayer of follicle cells remains on the oocyte surface. To achieve this, one forcep holds the stalk where the oocyte attaches to the ovarian tissue while the other forcep acts to tear the layers nearby the stalk in order to unwrap the oocyte (like peeling an orange). Damaged oocytes are discarded because they become sensitized for maturation. Oocytes with only one or two outer layers can be injected, but with difficulty. Oocytes obtained from collagenased ovarian tissue tend to swell, making it difficult for the oocytes to take up the injection volume. Thus, mechanically dissected oocytes have the advantage of being easier to inject and heal better after injection. Oocytes obtained during the spring months are more responsive and more synchronous in reaching maturation. During the winter season, oocytes may take 8-10 h to mature in response to progesterone treatment, while in the spring they may take 3-4 h. Similar results were observed in the assay of oncogenic ras. To obtain more responsive oocytes during the winter months, pregnant mare serum gonadotropin (PMSG, CalBiochem) may be used to sensitize the oocytes. Injection of 25 units of PMSG into the dorsal lymph sac of frogs 24 h before use will generate oocytes that mature in 2.5-3 h in response to progesterone treatment (3). If the above maturation time is shorter than 2.5 h, the risk of spontaneous maturation of untreated oocytes will be very high. Careful monitoring of the progesterone control is very important.
Injection The equipment necessary for microinjection of ras into immature oocytes is listed below: 1. Dissecting microscope, e.g., Wild M8 Zoom stereomicroscope 2. Illuminator, e.g., Dolan-Jenner, Model 190 fiber optic illuminator 3. Micromanipulator, e.g., Narishigi mm-33 4. Micromanipulator stand 5. Needle holder 6. Microburet--syringe type 7. Hamilton syringe, Luer tip, volume, 100 #1 8. Polyethylene tubing PZ-60 (Clay Adams)
9. 60% glycerol for filling syringe 10. Injection needle made from 30-#1 microcap capillary (Drummond Scientific Co., Broomall, PA 19008) 11. Custom-made steel spring for retracting the Hamilton syringe plunger Our instruments and techniques for oocyte microinjection are adopted from Gurdon (5). The preparation and calibration of the microinjection needle are shown in Fig. 1.
Description of oocyte microinjection. Oocytes are placed in a petri dish (or in a petri dish containing agar wells fitted to the size of an oocyte) under a dissecting stereomicroscope. A glass needle loaded with ras approaches the oocyte from the side, at an angle of 45 ° (Fig. 2). Forceps are placed on the opposite side to keep the oocyte immobile when the agar wells are not being used. The micromanipulator advances the needle to pierce the oocyte through the equatorial region and the needle tip should stop at the center of the sphere. Positive pressure is applied through the syringe, which is connected to the needle by polyethylene tubing. Fluid meniscus movement through the needle is monitored (a 1-mm displacement delivers 50 nl of sample). Negative pressure is applied to stop fluid movement through the retraction of the plunger by steel spring action. The needle is retracted from the oocyte and the injection is completed. This system is very reliable and is able to handle very small sample volumes. Commonly, 50 nl of sample is injected into each oocyte. There are more sophisticated instruments designed for oocyte injection, such as one recently available from Drummond Scientific Co. Preparation of ras for oocyte injection. The synthetic human e-H-ras gene is expressed in Escherichia coli and the recombinant protein purified (6). The protein is dialyzed extensively against buffer A (50 mM H-glycerol phosphate, 5 mM MgC12, pH 7.4). The sample is centrifuged for 1 min at 15,000g in an Eppendorf microcentrifuge to remove any particulate matter that could block fluid uptake into the injection needle. Observation and Timing of Germinal Vesicle Breakdown Modified Ringer's saline solution at 21°C provides an optimum condition for maturation after the oocyte receives a ras protein injection. The migration of the nucleus (GV) toward the surface of the animal pole and the dissolution of the nuclear membrane (referred to as germinal vesicle breakdown, GVBD) result in the appearance of a whitish spot at the pigmented animal pole (Fig. 2). To confirm the breakdown of the nuclear membrane, oocytes in question may be fixed with 5% trichloroaeetic acid for 5 min and split open for verification (7). Oncogenic ras at a dosage of 14 ng induces GVBD 612 h after injection, while this process using normal ras can take as long as 40 h for completion and requires a
Xenopus laevis OOCYTES AND ras PROTEINS (a)
317 Animal pole
Mlcroplpet
O_
0
Hand pull
lmm
Hand pull
Heat
Equatorial region GVBD
()
()
A ~ 0,3
F I G . 2. Schematic drawing of a Xenopus oocyte undergoing germinal vesicle breakdown (GVBD) upon microinjection of oncogenic ras. The micropipet containing the protein sample is inserted into the equatorial region of a Xenopus oocyte. GVBD is shown to occur after 8-10 h.
mm
(b)
T o quantitate the activity of ras, the sample must be serially diluted in buffer A. Each dilution is injected into 5-10 oocytes. T h e lowest dilution of injected ras t h a t elicits GVBD is indicative of the strength of the ras sample. A control in which 25 nl of buffer A is injected into oocytes
Heati coil
0.3 mrn -- _ -- 140mm TABLE
O.02mm
1
R e a c t i v a t i o n of H u m a n c-H-ras va112 P r o t e i n i n Mevalonate-Deprived Xenopus Oocytes Rubber stopper as weight
Expt
Actual calibration marks are made w i t h w a t e r insoluble ink and l rnm apart
£
60% Glycerol
Air
Sample is pushed out and moved a few calibration marks
Measure size of drop
F I G . 1. Construction and calibration of capillaries for microinjection. (a) The capillary pipet is heated in the center and hand-pulled. (b) Using a heating coil, the pipet tip is formed by applying a weight on one end during heating. Approximate dimensions of the different sections of the pipet are described. (c) Calibration of capillaries for fluid microinjection.
100-fold increase in dosage. In comparison, progesterone (2 #g/ml)-induced GVBD takes place 1-2 h earlier t h a n t h a t induced by oncogenic ras (4). Because partial response to GVBD is not well defined, GVBD in an oocyte is scored as an all or nothing event.
c-H-ras wu2 (ng)
Mevalonate (ng)
Oocytes with GVBD
1
0 0 0 0
56 28 14 7
0 0 0 0
6/6 6/6 6/6 0/6
2
12.5 1.25 0.125
0 0 0
0 0 0
0/6 O/6 O/6
3
12.5 1.25 0.125
56 56 56
0 0 0
0/6 0/6 0/6
4
0.125 0.125 0.125 0 0.125
28 28 28 28 0
4 40 400 0 0
0
0
4
0/3
0 0
0 0
40 400
0/3 0/3
(c) Polyethylentubing
Compactin (ng)
6/6* 6/6* 6/6" 3/3 0/3
Note. Oocyte injections were performed as described in the text. In all experiments a total volume of 50 nl was injected. Initiation of meiosis was measured visually by the appearance of germinal vesicle breakdown (GVBD), which occurred at 8 h after injection except in experiments labeled with an asterisk, in which GVBD occurred at 5 h after injection. In Experiments 3 and 4, oocytes were injected with compactin (25 nl) and t h e n incubated for 1 h at room temperature to achieve mevalonate deprivation and allow depletion of the isoprene pool; they were t h e n injected with h u m a n c-H-ras vau2 protein (25 nl) or a mixture of this protein and mevalonate (25 nl). Compactin was diluted from a stock solution (25 mg/ml) in 25% ethanol. For each experiment three to five oocytes were activated by progesterone treatment as a control for the ability of the oocytes from a particular frog to initiate meiosis (8).
318
WU, KIM, AND KIM
will rule out spontaneous GVBD induction. Table 1 lists results from experiments involving an oncogenic ras with a valine-for-glycine substitution of position 12 (c-HrasVan2); the drug compactin, which inhibits mevalonate synthesis; and mevalonate (8). GVBD induced by c-Hras w112 can be inhibited by compactin and this inhibition can be overcome by mevalonate, an intermediate of cholesterol biosynthesis. These results support the idea that a farnesyl moiety or its derivative is involved in the modification of the ras protein. Double injections can be given, but the total volume is kept at 50 nl or below.
DISCUSSION I
I
The ability of oncogenic ras to induce oocytes to enter meiosis, resulting in the breakdown of the nucleus, provides a fast and easy way to quantitate the activity of the protein, thus providing a convenient system for screening inhibitors or activators of ras protein under an in vivo condition before testing on an animal system. Oncogenic ras can also be injected into tissue culture cells to induce transformation to provide a qualitative study of its activity (9). The mechanism of ras-induced meiosis in oocytes is
not well understood. An attempt to compare it to progesterone induction showed that ras uses an alternate pathway to trigger meiosis in X e n o p u s oocytes that bypasses changes in intracellular cAMP levels (4). Further understanding of the induction of oocyte maturation by ras might help elucidate how this oncogenic protein functions in tumorigenesis.
REFERENCES I
I
I
1. Masui, Y. (1967) J. Exp. Zool. 166, 365-376. 2. Smith, L. D., Eeker, R. E., and Subtelney, S. (1968) Dev. Biol. 17, 627-643. 3. Smith, L. D. (1989) Development 107, 685-699. 4. Birchmeier, C., Broek, D., and Wiglet, M. (1985) Cell 43,615-621. 5. Gurdon, J. B. (1977) Methods Cell Biol. 16, 125-139.
6. Miura, K., Inoue, Y., Nakamori, H., Iwai, S., Ohtsuka, E., Ikehara, M., Noguchi, S., and Nishimura, S. (1986) Jpn. J. Cancer Res. 77, 45-51. 7. Trahey, M., and McCormick, F. (1987) Science 238, 542-545. 8. Schafer, W. R., Kim, R., Sterne, R., Thorner, J., Kim, S.-H., and Rine, J. (1989) Science 245, 379-385. 9. Feramisco, J. R., Kamata, T., Gross, M., Rosenberg, M., and Sweet, R. W. (1984) Cell 38, 107-117.