DEVELOPMENTAL
BIOLOGY
146,246-249
(1991)
Maturation-Promoting Factor and ~34~~~~Kinase during Oocyte Maturation of the Japanese Quail M. MORI,* M. YAMASHITA, Laboratory
of Reprodwtive *Depurtment
Biology, of Animal
M. YOSHIKUNI, National Science,
Accepted
Institute Shizuoka
S. FUKADA, AND Y. NAGAHAMA f&r Basic University,
March
Biology, Okazaki 444, Japan; Shizuoka 422, Japan
and
28, 1991
Maturation-promoting factor and a homolog of fission yeast c&2+ gene product (~34”~‘) were investigated during the final 24 hr of maturation of quail oocytes. Kinase activity of p34cdc2. m the oocyte germinal disk (GD) increased 15 times at maturation. Two bands, at 32 and 34 kDa, were detected in immature oocytes by immunoblotting of SDS-PAGE with anti-p34cd”2 monoclonal antibody. A new band, which is close to the 32-kDa band but with a slightly faster mobility, appeared during maturation. No ~34”~~ could be detected outside the GD. Microinjection of GD extract from mature 0 1991 Academic Press, Inc. oocytes caused maturation of Xenopus oocytes.
INTRODUCTION
Maturation-promoting factor (MPF), originally found during meiosis in frog oocytes (Masui and Markert, 1971), is a cytoplasmic factor, highly conserved among a wide range of species, that plays a key role in the progression of the cell cycle from interphase to metaphase, in both meiosis and mitosis (see review by Lohka, 1989). Recent progress in the characterization of MPF has clarified that one of the components of MPF is a 34-kDa protein encoded by the fission yeast, Schixosaccharomyces pombe, cd&+ gene (~34~~“‘) (Dunphy et al., 1988; Gautier et al., 1988; Labbk et al., 1989a, b). ~34”~“” can be detected immunologically by a polyclonal antibody raised against the PSTAIR sequence, the most conserved amino acid sequence of ~34”~~’ (Lee and Nurse, 1987). p34CdC”,as well as the highly purified MPF, contains a protein kinase activity, which can be measured by using histone Hl as an exogenous substrate (Lohka et al., 1988; Labbi! et al., 198913; see also review by Nurse, 1990). Avian oocyte maturation is faithfully regulated by the lighting conditions so that we can easily predict the precise time course of oocyte maturation. This is of great advantage in analyzing a maturation-inducing pathway in vivo, from an external stimulus to oocyte maturation. Actually several authors have succeeded in examining a surge of gonadotropin (luteinizing hormone) 6 hr before ovulation (Furr et al., 1973; Doi et ab, 1980), concomitant with the production of progesterone, the presumptive maturation-inducing hormone in birds, in follicle cells (Mori and Kantou, 1987). The first indications of germinal vesicle breakdown (GVBD) can be detected at 4.5 hr before ovulation and the first polar body 0012-1606/91 Copyright All rights
$3.00
#Zi 1991 by Academic Press, Inc. of reproduction in any form reserved.
246
is extruded between 2 and 4.5 hr before ovulation (Olsen and Fraps, 1950). On the other hand, avian oocytes are exceptionally large and extremely telolecithal. These characteristics have prevented detailed studies on the mechanisms of oocyte maturation within the oocytes. In fact, there are no reports on MPF from the oocytes of birds. This paper describes experiments on quail oocytes designed to detect the changes in ~34”~~’ and its kinase activity during oocyte maturation. The presence of MPF activity in quail oocytes is confirmed by microinjection into immature Xenopus oocytes. MATERIALS
AND
METHODS
Materials
Laying Japanese quail (Coturnix coturnix japonica), provided with food and water ad lib&urn, were caged individually on a lighting regime of 14 L:lO D, with lights on at 0500 hr. The time of ovulation was predicted based on the time of oviposition of the preceding egg, which usually occurred 15 to 30 min prior to the next ovulation (Woodard and Mather, 1964). To obtain maturing and mature oocytes, quails were killed 4 hr before ovulation, or just prior to ovulation, by decapitation and the largest follicles were isolated. To obtain immature oocytes 24 hr before ovulation, the second largest follicles also were isolated from the quails killed just prior to ovulation. After thecal cells were removed with fine forceps, 15 ~1 of oocyte cytoplasm was collected from the germinal disc (GD), which was identified as a small white spot on the surface of the yellow yolk. For con-
247
BRIEF NOTES
trols, the same amount of cytoplasm was collected separately from the periphery of the GD. Immediately after collection of the cytoplasm, it was diluted with 100 ~1 of extraction buffer (100 mM /3-glycerophosphate, 15 mM MgCl,, 5 mM EGTA, 1 mM dithiothreitol, 300 PLM (pamidinophenyl) methanesulfonyl fluoride, 3 pg/ml leupeptin, and 20 mM Hepes, pH 7.5). The mixture was centrifuged at 18,500g for 5 min at 4°C and the clear supernatant was used for kinase assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). Assay for p.?4cd’2 Kinase
[
non-GD
m
GD
Activity 0
p13”Uc1, the SUCI+ gene product of the fission yeast, binds to ~34”~“~ without inhibiting the kinase activity of P34cd”“. This binding is highly specific for p34cdc2, so that the kinase activity precipitated with ~13”“’ is almost completely attributable to ~34”~“‘(Pondaven et al., 1990). We used p13”““‘- Sepharose gel (9.8 mg p13=“‘/ml gel) to precipitate ~34~~~’ preferentially from the sample and measured the kinase activity bound to the gel. For the kinase assay, 75 ~1 of the supernatant was mixed with 10 pl of p13yUc’- Sepharose gel at 4°C overnight. The gel was washed three times with an excess of the extraction buffer and finally suspended in 20 ~1 of the same buffer. The kinase reaction was started by adding 30 ~1 of reaction buffer. The final concentration of the reaction mixture was 500 PLM ATP, 100 puM histone Hl (Type III-S; Sigma), 1.5 &i [y-32P]ATP (sp act; 6000 Ci/mmole), 1 mM EGTA, 10 mM MgCl,, 4.5 mM fl-mercaptoethanol, and 20 mM Tris-HCl (pH 7.4). After 10 min of incubation at 30°C the reaction was stopped by adding 5 ~1 of 3 M phosphoric acid, and the mixture was spotted on phosphocellulose paper (Whatman P81). The paper was washed five times with 1% phosphoric acid and once with acetone, and the remaining radioactivity was counted by a liquid scintillation spectrometer (Witt and Roskowski, 1975). For control incubation, untreated p13”““- Sepharose gel was used as an enzyme source. Electrophoresis
24
hr
HOURS
0 hr
OVULATION
FIG. 1. ~34’~’ kinase activity in quail oocytes. The extracts from germinal disk (GD) or from cytoplasm outside the germinal disk (nonGD) were prepared from oocytes at various times prior to ovulation. gel was assayed using The kinase activity bound to ~13~’ Sepharose histone Hl as substrate. The value of the control incubation, in which untreated p13=“- Sepharose gel was used as an enzyme source, was subtracted. Values are means t SEM of the number of oocytes in parentheses.
alized with 0.2 mM 5-bromo-4-chloro-3-indolyl phate and 0.2 mikf nitro blue tetrazolium. Assay fur MPF
phos-
Activity
In order to assess the MPF activity, 15 ~1 of GD was mixed with 10 yl of the extraction buffer and centrifuged at 18,500g for 5 min at 4°C. MPF activity was assayed by microinjecting 100 nl of the supernatant into a full-grown immature Xenopus oocyte in the presence of cycloheximide (20 pg/ml), according to the procedure of Wu and Gerhart (1980). GVBD was determined by the appearance of a white spot on the animal pole of the oocytes within 2 hr after injection and confirmed by dissecting the oocytes after boiling. RESULTS
and Immunoblotting p34cdc”
For SDS-PAGE, 25 ~1 of the supernatant was mixed with 5 ~1 of p13”““‘- Sepharose gel at 4°C overnight. After washing with the extraction buffer, the gel was boiled for 5 min in 10 ~1 of Laemmli sample buffer (Laemmli, 1970) and a 5+1 aliquot was loaded on a 12.5% polyacrylamide gel and separated at a constant current of 15 mA. After the proteins were transferred by electroblotting to Immobilon membrane (Millipore), was detected with a monoclonal antibody against P34’d’s the PSTAIR sequence (Yamaguchi et al., 1991). Antigen-antibody complexes were detected by alkaline phosphatase-conjugated goat anti-mouse IgG and visu-
4 hr BEFORE
Kinase
Activity
p34Cd”Z kinase activity in quail oocytes was measured during the final 24 hr of maturation (Fig. 1). The activity outside the GD remained low throughout the measured 24-hr period of maturation. Similar low activity was found in the GD of immature oocytes obtained from the second largest follicles, which were expected to ovulate 24 hr later. Oocytes from the largest follicles, removed 4 hr before the expected time of ovulation, had undergone GVBD and showed more than 15 times high activity in GD. The high activity in GD was also prominent in mature oocytes removed just prior to ovulation.
248
DEVELOPMENTAL BIOLOGY kD 67,
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20.1,
12
3
4
FIG. 2. Immunoblotting of extracts from quail oocytes with antiPSTAIR monoclonal antibody following SDS-PAGE. Asterisks show the 32- and 34-kDa bands, which are detected in germinal disk (GD) of immature oocytes. Note that a new band, which is close to the 32-kDa band but with a slightly faster mobility (arrow), appears after GVBD. Lane 1, control sample from cytoplasm outside the GD of an oocyte 4 hr before ovulation. Lane 2, GD of an immature oocyte 24 hr before ovulation. Lane 3, GD of maturing oocyte 4 hr before ovulation. Lane 4, GD of mature oocyte just prior to ovulation.
Immunoblotting
The proteins extracted from oocytes were separated on SDS-PAGE and immunoblotted with the antiPSTAIR monoclonal antibody (Fig. 2). Two bands, corresponding to 32 and 34 kDa, were detected in the extracts from GD of immature oocytes. A new band, which is close to the 32-kDa band but with a slightly faster mobility, was detected in the extracts from maturing and mature oocytes. No immunoreactive proteins were detected in the extract outside the GD. MPF Activity
Table 1 shows the results of microinjection experiments. There was a sixfold increase in the number of Xenopus oocytes maturing following injection of GD extracts from mature oocytes just prior to ovulation, compared with immature oocytes 24 hr before ovulation. DISCUSSION
This is the first report on the presence of MPF in avian oocytes. Recently, MPF has been purified from the oocytes of Xenopus (Lohka et al., 1988, Gautier et al., 1988) and starfish (Labbe et al., 1989a,b), and it has been demonstrated that ~34’~’ is a catalytic subunit of MPF (see also review by Nurse, 1990). The following findings strongly suggest that avian MPF, also, contains ~34”~’ as its catalytic subunit. (1) Avian MPF induced oocyte maturation in Xenopus. This finding supports the uni-
VOLUME 146. 1991
versal occurrence of highly conserved MPF activity in eukaryote cells. (2) The presence of ~34~~~ in avian oocytes was demonstrated by means of anti-PSTAIR monoclonal antibody, which recognizes ~34”~’ in fission yeast with high specificity (Yamaguchi et ab, 1991). (3) Presumptive active p34’&’ (see discussion below) appeared and the kinase activity of ~34”~~ increased in accordance with the appearance of MPF activity. MPF exists in an inactive form in immature oocytes (Dunphy and Newport, 1988). Under the influence of gonadotropin, maturation-inducing hormone activates MPF, and this process is accompanied by dephosphorylation of ~34’~‘” (see review by Nurse, 1990). We have shown that avian oocytes have ~34”~’ activity which migrates as three different bands on one-dimensional SDS-PAGE and that the fastest migrating form appeared only after GVBD. Three forms of ~34”~’ have also been found in mammalian cell cultures, and the fastest migrating form has been shown to be dephosphorylated ~34”~~ (Draetta and Beach, 1988; Morla et al., 1989). It is therefore highly likely that the fastest migrating form of ~34”~’ detected only after GVBD in avian oocytes is the dephosphorylated, active form of p34cdc2. The findings, reported here, that extracts from quail immature oocytes have a low level of MPF activity, may be caused by cyclins, which are another component of MPF (Gautier et al., 1988; Labbe et al, 1989a). It has been reported that introduction of a high quantity of cyclins, by injection of cyclin mRNA, induces oocyte maturation in Xenopus (Westendorf et al., 1989). In addition, cyclins are known to be present even in immature oocytes of et ah, 1989) and Xenopus (Gautier clam (Westerndorf and Maller, 1991). Quail immature oocytes may contain a relatively high quantity of cyclins, so that its injection may cause oocytes maturation in Xenopus with low frequency. Birds provide a good system for analyzing a maturation-inducing pathway, because we can predict the pre-
TABLE 1 QUAIL OOCYTE MPF ACTIVITY, EXPRESSED AS FREQUENCY OF GVBD IN IMMATURE XENOPUS OOCYTES~ GVBDb Injected material
A
B
Extraction buffer Immature oocyte extract Mature oocyte extract
O/12
o/19
5/34
l/12
15/20
702
Total 0% 11.5% 66.6%
a Each material was injected into two batches (A and B) of Xenopus oocytes (refer to Materials and Methods). b Number of oocytes showing GVBD/number of ooeytes injected.
249
BRIEF NOTES
cise time course of oocyte maturation in vivo (see the introduction). We have shown that avian oocytes are also useful for studies on the mechanisms of oocyte maturation within the oocytes, by demonstrating the presence of MPF and ~34~~” kinase. We believe that aves are one of the best models to analyze the whole process of oocyte maturation, from an external stimulus (lighting) to MPF, via gonadotropin and maturation-inducing hormone.
(1989a). MPF from starfish oocytes at first meiotic metaphase is a heterodimer containing one molecule of cd& and one molecule of cyclin B. EMBO J. 8,3053-3058. LABB$, J.-C., PICARD, A., PEAUCELLIER, G., CAVADORE, J.-C., NURSE, P., and DOR$E, M. (1989b). Purification of MPF from starfish: Identification as the Hl histone kinase ~34’” and a possible mechanism for its periodic activation. Cell 57,253-263. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680-685. LEE, M. G., and NURSE, P. (1987). Complementation used to clone a human homologue of the fission yeast cell cycle control gene cd&.
We are grateful to Dr. P. Nurse for critical reading of the manuscript and to Dr. J. Hayles who prepared the bacterial strain overexpressing ~13~‘. We also thank Dr. I. G. Gleadall for helpful comments on the manuscript. This research was in part supported by Grants-inAid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (02102010 to Y.N.), the Naito Foundation, and the Japan Health Sciences Foundation.
LOHKA, M. J. (1989). Mitotic control by metaphase-promoting factor and cdc proteins. J. Cell Sci. 92, 131-135. LOHKA, M. J., HAYES, M. K., and MALLER, J. L. (1988). Purification of maturation-promoting factor, an intracellular regulator of early mitotic events. Proc. Natl. Acad. Sci. USA 85,3009-3013. MASUI, Y., and MARKERT, C. L. (1971). Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J. Exp. Zoo1 177,129-146. MORI, M., and KANTOU, T. (1987). Changes in progesterone production in granulosa cells during the ovulatory cycle of the Japanese quail (Coturnix coturnix jupmica). Gen. Camp. Endocrinol. 68, 57-63. MORLA, A. O., DRAETTA, G., BEACH, D., and WANG, J. Y. J. (1989). Reversible tyrosine phosphorylation of c&Z: Dephosphorylation accompanies activation during entry into mitosis. Cell 58, 193-203. NURSE, P. (1990). Universal control mechanism regulating onset of M-phase. Nature 344,503-508. OLSEN, M. W., and FRAPS, R. M. (1950). Maturation changes in the hen’s ovum. J. Exp. 2001. 114,475-489. PONDAVEN, P., MEIJER, L., and BEACH, D. (1990). Activation of Mphase-specific histone Hl kinase by modification of the phosphorylation of its ~34”~’ and cyclin components. Gene De?,. 4.9917. WESTENDORF, J. M., SWENSON,K. I., and RUDERMAN, J. V. (1989). The role of cyclin B in meiosis I. J. Cell Biol. 108, 1431-1444. WITT, J. J., and ROSKOWSKI, R. (1975). Rapid protein kinase assay using phosphocellulose-paper absorption. Anul. Biochum. 66, 253258. WOODARD, A. E., and MATHER, F. B. (1964). The timing of ovulation, movement of the ovum through the oviduct, pigmentation and shell deposition in Japanese quail (Coturmiz cotumixjapmica). Poult. Sri. 43,1427-1432. WU, M., and GERHART, J. C. (1980). Partial purification and characterization of the maturation-promoting factor from eggs of Xenm
Nature
REFERENCES DOI, O., TAKAI, T., NAKAMURA, T., and TANABE, Y. (1980). Changes in the pituitary and plasma LH, plasma and follicular progesterone and estradiol, and plasma testosterone and estrone concentrations during the ovulatory cycle of the quail (Cotur& coturnizjapmzica). Gen. Comp. Endocrinol. 41, 156-163. DRAE~A, G., and BEACH, D. (1988). Activation of c&Z protein kinase during mitosis in human cells: Cell cycle-dependent phosphorylation and subunit rearrangement. Cell 54,17-26. DUNPHY, W. G., BRIZUELA, L., BEACH, D., and NEWPORT, J. (1988). The Xenopus cd& protein is a component of MPF, a cytoplasmic regulator of mitosis. Cell 54, 423-431. DUNPHY, W. G., and NEWPORT, J. W. (1988). Mitosis-inducing factors are present in a latent form during interphase in the Xenopua embryo. J. Cell Biol. 106, 2047-2056. FURR, R. J. A., BONNEY, R. C., ENGLAND, R. J., and CUNNINGHAM, F. J. (1973). Luteinizing hormone and progesterone in peripheral blood during the ovulatory cycle of the hen Gallus domesticus. J. End@ crinol. 57, 159-169. GAUTIER, J., and MALLER, J. (1991). Cyclin B in Xenopus oocytes: Implications for the mechanisms of pre-MPF activation. EMBO J. 10, 177-182. GAUTIER, J., NORBURY, C., LOHKA, M., NURSE, P., and MALLER, J. (1988). Purified maturation-promoting factor contains the product of a Xenopus homolog of the fission yeast cell cycle control gene cdc2’.
Cell 54,433-439.
LABB~, J.-C., CAPONY, J.-P., CAPUT, D., CAVADORE, J.-C., DERANCOURT, J., KAGHAD, M., LELIAS, J.-M., PICARD, A., and DOR~E, M.
lueuis.
327,31-35.
Den. Biol.
79, 465-477.
YAMAGUCHI, A., YAMASHITA, M., YOSHIKUNI, M., Ho~A, Y., NURSE, P., and NAGAHAMA, Y. (1991). Involvement in meiotic prophase of HI histone kinase, ~34”~” homologues and maturation-promoting factor (MPF) in lily (Lillium lwngijbum) microsporocytes. Submitted for publication.
BIOLOGY
146,246-249
(1991)
Maturation-Promoting Factor and ~34~~~~Kinase during Oocyte Maturation of the Japanese Quail M. MORI,* M. YAMASHITA, Laboratory
of Reprodwtive *Depurtment
Biology, of Animal
M. YOSHIKUNI, National Science,
Accepted
Institute Shizuoka
S. FUKADA, AND Y. NAGAHAMA f&r Basic University,
March
Biology, Okazaki 444, Japan; Shizuoka 422, Japan
and
28, 1991
Maturation-promoting factor and a homolog of fission yeast c&2+ gene product (~34”~‘) were investigated during the final 24 hr of maturation of quail oocytes. Kinase activity of p34cdc2. m the oocyte germinal disk (GD) increased 15 times at maturation. Two bands, at 32 and 34 kDa, were detected in immature oocytes by immunoblotting of SDS-PAGE with anti-p34cd”2 monoclonal antibody. A new band, which is close to the 32-kDa band but with a slightly faster mobility, appeared during maturation. No ~34”~~ could be detected outside the GD. Microinjection of GD extract from mature 0 1991 Academic Press, Inc. oocytes caused maturation of Xenopus oocytes.
INTRODUCTION
Maturation-promoting factor (MPF), originally found during meiosis in frog oocytes (Masui and Markert, 1971), is a cytoplasmic factor, highly conserved among a wide range of species, that plays a key role in the progression of the cell cycle from interphase to metaphase, in both meiosis and mitosis (see review by Lohka, 1989). Recent progress in the characterization of MPF has clarified that one of the components of MPF is a 34-kDa protein encoded by the fission yeast, Schixosaccharomyces pombe, cd&+ gene (~34~~“‘) (Dunphy et al., 1988; Gautier et al., 1988; Labbk et al., 1989a, b). ~34”~“” can be detected immunologically by a polyclonal antibody raised against the PSTAIR sequence, the most conserved amino acid sequence of ~34”~~’ (Lee and Nurse, 1987). p34CdC”,as well as the highly purified MPF, contains a protein kinase activity, which can be measured by using histone Hl as an exogenous substrate (Lohka et al., 1988; Labbi! et al., 198913; see also review by Nurse, 1990). Avian oocyte maturation is faithfully regulated by the lighting conditions so that we can easily predict the precise time course of oocyte maturation. This is of great advantage in analyzing a maturation-inducing pathway in vivo, from an external stimulus to oocyte maturation. Actually several authors have succeeded in examining a surge of gonadotropin (luteinizing hormone) 6 hr before ovulation (Furr et al., 1973; Doi et ab, 1980), concomitant with the production of progesterone, the presumptive maturation-inducing hormone in birds, in follicle cells (Mori and Kantou, 1987). The first indications of germinal vesicle breakdown (GVBD) can be detected at 4.5 hr before ovulation and the first polar body 0012-1606/91 Copyright All rights
$3.00
#Zi 1991 by Academic Press, Inc. of reproduction in any form reserved.
246
is extruded between 2 and 4.5 hr before ovulation (Olsen and Fraps, 1950). On the other hand, avian oocytes are exceptionally large and extremely telolecithal. These characteristics have prevented detailed studies on the mechanisms of oocyte maturation within the oocytes. In fact, there are no reports on MPF from the oocytes of birds. This paper describes experiments on quail oocytes designed to detect the changes in ~34”~~’ and its kinase activity during oocyte maturation. The presence of MPF activity in quail oocytes is confirmed by microinjection into immature Xenopus oocytes. MATERIALS
AND
METHODS
Materials
Laying Japanese quail (Coturnix coturnix japonica), provided with food and water ad lib&urn, were caged individually on a lighting regime of 14 L:lO D, with lights on at 0500 hr. The time of ovulation was predicted based on the time of oviposition of the preceding egg, which usually occurred 15 to 30 min prior to the next ovulation (Woodard and Mather, 1964). To obtain maturing and mature oocytes, quails were killed 4 hr before ovulation, or just prior to ovulation, by decapitation and the largest follicles were isolated. To obtain immature oocytes 24 hr before ovulation, the second largest follicles also were isolated from the quails killed just prior to ovulation. After thecal cells were removed with fine forceps, 15 ~1 of oocyte cytoplasm was collected from the germinal disc (GD), which was identified as a small white spot on the surface of the yellow yolk. For con-
247
BRIEF NOTES
trols, the same amount of cytoplasm was collected separately from the periphery of the GD. Immediately after collection of the cytoplasm, it was diluted with 100 ~1 of extraction buffer (100 mM /3-glycerophosphate, 15 mM MgCl,, 5 mM EGTA, 1 mM dithiothreitol, 300 PLM (pamidinophenyl) methanesulfonyl fluoride, 3 pg/ml leupeptin, and 20 mM Hepes, pH 7.5). The mixture was centrifuged at 18,500g for 5 min at 4°C and the clear supernatant was used for kinase assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). Assay for p.?4cd’2 Kinase
[
non-GD
m
GD
Activity 0
p13”Uc1, the SUCI+ gene product of the fission yeast, binds to ~34”~“~ without inhibiting the kinase activity of P34cd”“. This binding is highly specific for p34cdc2, so that the kinase activity precipitated with ~13”“’ is almost completely attributable to ~34”~“‘(Pondaven et al., 1990). We used p13”““‘- Sepharose gel (9.8 mg p13=“‘/ml gel) to precipitate ~34~~~’ preferentially from the sample and measured the kinase activity bound to the gel. For the kinase assay, 75 ~1 of the supernatant was mixed with 10 pl of p13yUc’- Sepharose gel at 4°C overnight. The gel was washed three times with an excess of the extraction buffer and finally suspended in 20 ~1 of the same buffer. The kinase reaction was started by adding 30 ~1 of reaction buffer. The final concentration of the reaction mixture was 500 PLM ATP, 100 puM histone Hl (Type III-S; Sigma), 1.5 &i [y-32P]ATP (sp act; 6000 Ci/mmole), 1 mM EGTA, 10 mM MgCl,, 4.5 mM fl-mercaptoethanol, and 20 mM Tris-HCl (pH 7.4). After 10 min of incubation at 30°C the reaction was stopped by adding 5 ~1 of 3 M phosphoric acid, and the mixture was spotted on phosphocellulose paper (Whatman P81). The paper was washed five times with 1% phosphoric acid and once with acetone, and the remaining radioactivity was counted by a liquid scintillation spectrometer (Witt and Roskowski, 1975). For control incubation, untreated p13”““- Sepharose gel was used as an enzyme source. Electrophoresis
24
hr
HOURS
0 hr
OVULATION
FIG. 1. ~34’~’ kinase activity in quail oocytes. The extracts from germinal disk (GD) or from cytoplasm outside the germinal disk (nonGD) were prepared from oocytes at various times prior to ovulation. gel was assayed using The kinase activity bound to ~13~’ Sepharose histone Hl as substrate. The value of the control incubation, in which untreated p13=“- Sepharose gel was used as an enzyme source, was subtracted. Values are means t SEM of the number of oocytes in parentheses.
alized with 0.2 mM 5-bromo-4-chloro-3-indolyl phate and 0.2 mikf nitro blue tetrazolium. Assay fur MPF
phos-
Activity
In order to assess the MPF activity, 15 ~1 of GD was mixed with 10 yl of the extraction buffer and centrifuged at 18,500g for 5 min at 4°C. MPF activity was assayed by microinjecting 100 nl of the supernatant into a full-grown immature Xenopus oocyte in the presence of cycloheximide (20 pg/ml), according to the procedure of Wu and Gerhart (1980). GVBD was determined by the appearance of a white spot on the animal pole of the oocytes within 2 hr after injection and confirmed by dissecting the oocytes after boiling. RESULTS
and Immunoblotting p34cdc”
For SDS-PAGE, 25 ~1 of the supernatant was mixed with 5 ~1 of p13”““‘- Sepharose gel at 4°C overnight. After washing with the extraction buffer, the gel was boiled for 5 min in 10 ~1 of Laemmli sample buffer (Laemmli, 1970) and a 5+1 aliquot was loaded on a 12.5% polyacrylamide gel and separated at a constant current of 15 mA. After the proteins were transferred by electroblotting to Immobilon membrane (Millipore), was detected with a monoclonal antibody against P34’d’s the PSTAIR sequence (Yamaguchi et al., 1991). Antigen-antibody complexes were detected by alkaline phosphatase-conjugated goat anti-mouse IgG and visu-
4 hr BEFORE
Kinase
Activity
p34Cd”Z kinase activity in quail oocytes was measured during the final 24 hr of maturation (Fig. 1). The activity outside the GD remained low throughout the measured 24-hr period of maturation. Similar low activity was found in the GD of immature oocytes obtained from the second largest follicles, which were expected to ovulate 24 hr later. Oocytes from the largest follicles, removed 4 hr before the expected time of ovulation, had undergone GVBD and showed more than 15 times high activity in GD. The high activity in GD was also prominent in mature oocytes removed just prior to ovulation.
248
DEVELOPMENTAL BIOLOGY kD 67,
43,
20.1,
12
3
4
FIG. 2. Immunoblotting of extracts from quail oocytes with antiPSTAIR monoclonal antibody following SDS-PAGE. Asterisks show the 32- and 34-kDa bands, which are detected in germinal disk (GD) of immature oocytes. Note that a new band, which is close to the 32-kDa band but with a slightly faster mobility (arrow), appears after GVBD. Lane 1, control sample from cytoplasm outside the GD of an oocyte 4 hr before ovulation. Lane 2, GD of an immature oocyte 24 hr before ovulation. Lane 3, GD of maturing oocyte 4 hr before ovulation. Lane 4, GD of mature oocyte just prior to ovulation.
Immunoblotting
The proteins extracted from oocytes were separated on SDS-PAGE and immunoblotted with the antiPSTAIR monoclonal antibody (Fig. 2). Two bands, corresponding to 32 and 34 kDa, were detected in the extracts from GD of immature oocytes. A new band, which is close to the 32-kDa band but with a slightly faster mobility, was detected in the extracts from maturing and mature oocytes. No immunoreactive proteins were detected in the extract outside the GD. MPF Activity
Table 1 shows the results of microinjection experiments. There was a sixfold increase in the number of Xenopus oocytes maturing following injection of GD extracts from mature oocytes just prior to ovulation, compared with immature oocytes 24 hr before ovulation. DISCUSSION
This is the first report on the presence of MPF in avian oocytes. Recently, MPF has been purified from the oocytes of Xenopus (Lohka et al., 1988, Gautier et al., 1988) and starfish (Labbe et al., 1989a,b), and it has been demonstrated that ~34’~’ is a catalytic subunit of MPF (see also review by Nurse, 1990). The following findings strongly suggest that avian MPF, also, contains ~34”~’ as its catalytic subunit. (1) Avian MPF induced oocyte maturation in Xenopus. This finding supports the uni-
VOLUME 146. 1991
versal occurrence of highly conserved MPF activity in eukaryote cells. (2) The presence of ~34~~~ in avian oocytes was demonstrated by means of anti-PSTAIR monoclonal antibody, which recognizes ~34”~’ in fission yeast with high specificity (Yamaguchi et ab, 1991). (3) Presumptive active p34’&’ (see discussion below) appeared and the kinase activity of ~34”~~ increased in accordance with the appearance of MPF activity. MPF exists in an inactive form in immature oocytes (Dunphy and Newport, 1988). Under the influence of gonadotropin, maturation-inducing hormone activates MPF, and this process is accompanied by dephosphorylation of ~34’~‘” (see review by Nurse, 1990). We have shown that avian oocytes have ~34”~’ activity which migrates as three different bands on one-dimensional SDS-PAGE and that the fastest migrating form appeared only after GVBD. Three forms of ~34”~’ have also been found in mammalian cell cultures, and the fastest migrating form has been shown to be dephosphorylated ~34”~~ (Draetta and Beach, 1988; Morla et al., 1989). It is therefore highly likely that the fastest migrating form of ~34”~’ detected only after GVBD in avian oocytes is the dephosphorylated, active form of p34cdc2. The findings, reported here, that extracts from quail immature oocytes have a low level of MPF activity, may be caused by cyclins, which are another component of MPF (Gautier et al., 1988; Labbe et al, 1989a). It has been reported that introduction of a high quantity of cyclins, by injection of cyclin mRNA, induces oocyte maturation in Xenopus (Westendorf et al., 1989). In addition, cyclins are known to be present even in immature oocytes of et ah, 1989) and Xenopus (Gautier clam (Westerndorf and Maller, 1991). Quail immature oocytes may contain a relatively high quantity of cyclins, so that its injection may cause oocytes maturation in Xenopus with low frequency. Birds provide a good system for analyzing a maturation-inducing pathway, because we can predict the pre-
TABLE 1 QUAIL OOCYTE MPF ACTIVITY, EXPRESSED AS FREQUENCY OF GVBD IN IMMATURE XENOPUS OOCYTES~ GVBDb Injected material
A
B
Extraction buffer Immature oocyte extract Mature oocyte extract
O/12
o/19
5/34
l/12
15/20
702
Total 0% 11.5% 66.6%
a Each material was injected into two batches (A and B) of Xenopus oocytes (refer to Materials and Methods). b Number of oocytes showing GVBD/number of ooeytes injected.
249
BRIEF NOTES
cise time course of oocyte maturation in vivo (see the introduction). We have shown that avian oocytes are also useful for studies on the mechanisms of oocyte maturation within the oocytes, by demonstrating the presence of MPF and ~34~~” kinase. We believe that aves are one of the best models to analyze the whole process of oocyte maturation, from an external stimulus (lighting) to MPF, via gonadotropin and maturation-inducing hormone.
(1989a). MPF from starfish oocytes at first meiotic metaphase is a heterodimer containing one molecule of cd& and one molecule of cyclin B. EMBO J. 8,3053-3058. LABB$, J.-C., PICARD, A., PEAUCELLIER, G., CAVADORE, J.-C., NURSE, P., and DOR$E, M. (1989b). Purification of MPF from starfish: Identification as the Hl histone kinase ~34’” and a possible mechanism for its periodic activation. Cell 57,253-263. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680-685. LEE, M. G., and NURSE, P. (1987). Complementation used to clone a human homologue of the fission yeast cell cycle control gene cd&.
We are grateful to Dr. P. Nurse for critical reading of the manuscript and to Dr. J. Hayles who prepared the bacterial strain overexpressing ~13~‘. We also thank Dr. I. G. Gleadall for helpful comments on the manuscript. This research was in part supported by Grants-inAid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (02102010 to Y.N.), the Naito Foundation, and the Japan Health Sciences Foundation.
LOHKA, M. J. (1989). Mitotic control by metaphase-promoting factor and cdc proteins. J. Cell Sci. 92, 131-135. LOHKA, M. J., HAYES, M. K., and MALLER, J. L. (1988). Purification of maturation-promoting factor, an intracellular regulator of early mitotic events. Proc. Natl. Acad. Sci. USA 85,3009-3013. MASUI, Y., and MARKERT, C. L. (1971). Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J. Exp. Zoo1 177,129-146. MORI, M., and KANTOU, T. (1987). Changes in progesterone production in granulosa cells during the ovulatory cycle of the Japanese quail (Coturnix coturnix jupmica). Gen. Camp. Endocrinol. 68, 57-63. MORLA, A. O., DRAETTA, G., BEACH, D., and WANG, J. Y. J. (1989). Reversible tyrosine phosphorylation of c&Z: Dephosphorylation accompanies activation during entry into mitosis. Cell 58, 193-203. NURSE, P. (1990). Universal control mechanism regulating onset of M-phase. Nature 344,503-508. OLSEN, M. W., and FRAPS, R. M. (1950). Maturation changes in the hen’s ovum. J. Exp. 2001. 114,475-489. PONDAVEN, P., MEIJER, L., and BEACH, D. (1990). Activation of Mphase-specific histone Hl kinase by modification of the phosphorylation of its ~34”~’ and cyclin components. Gene De?,. 4.9917. WESTENDORF, J. M., SWENSON,K. I., and RUDERMAN, J. V. (1989). The role of cyclin B in meiosis I. J. Cell Biol. 108, 1431-1444. WITT, J. J., and ROSKOWSKI, R. (1975). Rapid protein kinase assay using phosphocellulose-paper absorption. Anul. Biochum. 66, 253258. WOODARD, A. E., and MATHER, F. B. (1964). The timing of ovulation, movement of the ovum through the oviduct, pigmentation and shell deposition in Japanese quail (Coturmiz cotumixjapmica). Poult. Sri. 43,1427-1432. WU, M., and GERHART, J. C. (1980). Partial purification and characterization of the maturation-promoting factor from eggs of Xenm
Nature
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