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Patterns of polypeptide synthesis in mouse oocytes during germinal vesicle breakdown and during maintenance of the germinal vesicle stage by dibutyryl cAMP
DEVELOPMENTALBIOLOGY83, 188-192 (1981)
Patterns of Polypeptide Synthesis in Mouse Oocytes during Germinal Vesicle Breakdown and during Maintenance of the Germinal Vesicle Stage by Dibutyryl cAMP JOEL D. RICHTER 1 A N D R O B E R T W. M C G A U G H E Y Department of Zoology, Arizona State University, Tempe, Arizona 85~81 Received June 19, 1980; accepted in revised farm August 18, 1980 Mouse oocytes undergo spontaneous meiotic maturation when cultured in vitro in the absence, but not in the presence, of dibutyryl cyclic AMP (cAMP). Furthermore, the inhibition by dibutyryl cAMP is reversible. Oocytes synthesize stage-specific polypeptides (i.e., developmental polypeptides) during meiotic maturation. Dibutyryl cAMP affects the synthesis of four of these developmental polypeptides by reversibly inhibiting the synthesis of one polypeptide, and irreversibly inhibiting the synthesis of three other polypeptides. Dibutyryl cAMP also reversibly inhibits the synthesis of at least two specific nondevelopmental polypeptides. INTRODUCTION
Most oocytes of the sexually mature mammal are arrested at late diplotene, a nuclear stage called the germinal vesicle (gv). In response to cyclical release of gonadotropins, some oocytes resume meiosis and undergo the first meiotic division in preparation for ovulation and fertilization. This developmental process, called maturation, will take place in mammalian oocytes which have been isolated from their follicles and maintained in culture (for review, see Donahue, 1972; Masui and Clarke, 1979). Oocyte maturation in vivo and spontaneous maturation in vitro involve not only chromosomal events, but also coordinated changes in synthesis of RNA (Wassarman and Letourneau, 1976) and of protein (McGaughey and Van Blerkom, 1977; Schultz and Wassarman, 1977a; Van Blerkom and McGaughey, 1978a). The regulation of mammalian oocyte maturation has been the subject of many recent studies. Several substances have been reported as potential regulators of oocyte maturation on the basis of their inhibition of spontaneous gv breakdown (gvb) in vitro (Cho et al., 1974; Tsafriri and Channing, 1975; McGaughey, 1977). One of these substances, dibutyryl cyclic adenosine monophosphate (dbcAMP), has been shown to block spontaneous maturation reversibly in oocytes from mice (Cho et al., 1974), rats (Dekel and Beers, 1978), and pigs (Rice and McGaughey, 1980). There is evidence that cyclic nucleotides and protein kinases perform regulatory functions during oocyte 1 Present address: Purdue University, Department of Biological Sciences, West Lafayette, Ind. 47907. 0012-1606/81/050188-05502.00/0 Copyright 9 1981by AcademicPress, Inc. All rights of reproductionin any form reserved.
188
maturation in amphibians (Speaker and Butcher, 1977; Morrill et a/., 1977; Mailer et al., 1977; Schorderet-Slatkin et a/., 1978; Mailer and Krebs, 1977). The mechanism by which dbcAMP acts to block gvb in mammalian oocytes is not known. However, we recently observed that although dbcAMP did not influence measurably the uptake of ~PO42-, its incorporation into phosphoprotein of mouse oocytes was decreased by dbcAMP relative to controls (Richter and McGaughey, in preparation). Since this apparent inhibition of protein phosphorylation may reflect a fundamental role for cAMP in the regulation of maturation in mammals, it has become essential that the influence of the analog on additional developmental parameters be examined. In this study we provide evidence that in the presence of dbcAMP, cultured mouse oocytes exhibit altered patterns of polypeptide synthesis. Our results can be interpreted, in part, to reflect a Potential regulatory function for cAMP in mammalian oocyte maturation. On the other hand, some of the molecular changes were not reversible upon removal of oocytes from the presence of dbcAMP, and therefore may reflect an abnormal, pharmacological influence of the analog on the subsequent development of mouse oocytes. MATERIALS AND METHODS
Oocytes and culture conditions. Oocytes from randomly bred young adult Swiss albino mice were harvested by puncturing ovarian follicles in minimal medium (Brinster, 1971) containing no dbcAMP (control medium) or 100 ~g/ml dbcAMP (Sigma). This dose was chosen as the lowest concentration at which maturation is reversibly inhibited by at least 90% (Cho et al., 1974).
BRIEF NOTES pH Z5
189 pH 43
IO ~= X
b-
.J
0 3E
23-
13FIG. 1. Autoradiogram of a high resolution two-dimensional polypeptide pattern of mouse oocytes. This sample contained 61 oocytes (476,000 cpm of TCA-precipitable radioactivity), which had undergone gvb during culture in control medium for 6 hr, the final 2 hr in the presence of [~S]methionine. Brackets designate areas of the autoradiogram in which the majority of changes in polypeptide patterns were observed. These areas are displayed in detail in the following figure.
Only oocytes which appeared normal under a dissection microscope and which had an intact gv were selected. Oocytes were cultured in 100-~1 drops of either control medium or medium with dbcAMP, in a humidified atmosphere of 5% CO2,5% 02, and 90% N2 at 37~ for 0, 2, or 4 hr, and subsequently for an additional 2 hr in the same medium but containing [35S]methionine (1.06 mCi/ml; spac, 840 Ci/mmole; Amersham). Other oocytes were cultured for a total of 18 hr: 12 hr in the presence of dbcAMP and 6 h r in control medium, the last 2 hr of which were in the radioisotope. Approximately 80% of the oocytes underwent gvb between 2 and 4 hr of culture in control medium. Oocytes which underwent early gvb in the 0- to 2-hr period and those which underwent gvb during the 4- to 6-hr period (with or without an initial 12-hr culture in dbcAMP) were excluded. None of the oocytes from the 2- to 4-hr period was removed. Culture medium with dbcAMP prevented gvb in approximately 95% of the oocytes. Oocytes which did not exhibit an intact gv during any of the culture periods in the presence of dbcAMP were excluded.
Two-dimensional polyacrylamide gel electrophoresis. Labeled oocytes were washed in radioinert medium and placed in 30 ~l of lysis buffer (O'Farrell, 1975) followed by repeated freezing and tha~viag. A 2- ~1 aliquot of the lysate was precipitated in TCA to estimate incorporation of [s5S]methionine. Two-dimensional electrophoresis was performed essentially as described by O'Farrell (1975), except that a linear gradient of 8 to 15% acrylamide was used in the second dimension. Exposures for autoradiograms followed those of Van Blerkom (1978).
Three different atitoradiograms were examined for each culture condition and period. RESULTS AND DISCUSSION
Diburyryl cAMP, but not butyric acid, inhibits mouse oocyte maturation (Cho et al., 1974), and neither butyrate nor cAMP exactly mimic the inhibitory effect of dbcAMP on rat oocytes (Dekel and Beers, 1980). Oocytes cultured for 12 hr in the presence of dbcAMP resume maturation when subsequently cultured in control medium and these reversed oocytes appear to be morphologically normal. We present evidence here that polypeptide synthesis is influenced by dbcAMP but that these molecular alterations are not all reversible. The autoradiograms from high-resolution polyacrylamide gel electrophoresiS were examined within and between experimental groups and were highly consistent for each type of sample. Reproducible changes in patterns of polypeptide synthesis were observed for control oocytes sampled before, during, and after, gvb a n d many of these changes were altered for oocytes in the presence of dbcAMP. A representative au~radiogram (Fig. 1)and portions of autoradiograms (Fig. 2) demonstrate the patterns and changes in patterns which we observed. The brackets in Fig. 1 designate areas in which the majority of changes were identified,and these are displayed in Fig. 2. A distinct progression of changes in pattern occurs during early maturation of control oocytes. Putative developmental polypeptides are shown in Figs. 2A (ar-
190
DEVELOPMENTAL BIOLOGY 0--2
HR
A
2 -- 4
HR
VOLUME83, 1981 4--6
HR
REV.
i 84 9
m
B
"1-
m
C
D
+
FIG. 2. (A) Comparison of area A (Fig. 1) from different autoradiograms. In this and subsequent figures 0-2, 2-4, and 4-6 hr refer to radiolabeling periods following culture in medium without ( - ) or with (+) 100/~g/ml dbcAMP. Reversal (REV) refers to polypeptide patterns during 4-6 hr of culture in control medium after initial culture for 12 hr in medium with dbcAMP. The incorporated radioactivity and numbers of oocytes for the samples in this and subsequent figures were:0-2, (305,000 cpm; 84 oocytes); 2-4 (366,000 cpm; 75 oocytes), 4-6
BRIEF NOTES
row), B (polypeptides 1 and 2), C (arrow), and D (polypeptide 3). In all five cases, these polypeptides were undetectable at 0-2 or 0-4 hr of culture, and were detectable for the first time during or following gvb. These developmental changes are of the same magnitude as those observed in rabbit (Van Blerkom and McGaughey, 1978a) and pig (McGaughey and Van Blerkom, 1977) oocytes during early stages of maturation. However, the synthesis of polypeptides peculiar to the period prior to gvb was not observed, unlike immature oocytes of the pig (McGaughey and Van Blerkom, 1977) and rabbit (Van Blerkom and McGaughey, 1978a). The synthetic patterns of the above developmental polypeptides were influenced in various ways in oocytes in which gvb was inhibited by dbcAMP. One of these polypeptides (Fig. 2A, arrow) exhibited an apparently normal pattern of synthesis (i.e., absent at 0-2hr, present at 2-6 hr) in the absence and presence of dbcAMP. Three (Fig. 2B, Nos. 1 and 2; Fig. 2D No. 3) were not detected in the presence of dbcAMP, although they were synthesized by control oocytes at 2-6 hr of culture. The synthesis of the fifth developmental polypeptide (Fig. 2C, arrow) was inhibited by dbcAMP during 2-6 hr of culture relative to controls; however, surprisingly this polypeptide was detectable at 0-2 hr of culture in the presence of dbcAMP, but not in controls. Reversal of inhibited synthesis for developmental polypeptides was observed in only one case (Fig. 2C, arrow). In the three other cases, polypeptides not detectable in samples of oocytes cultured in dbcAMP also were not detected in autoradiograms from oocytes undergoing reversal of gvb inhibition. The synthesis of two other polypeptides was inhibited reversibly in oocytes cultured in dbcAMP (Fig. 2D, Nos. 1 and 2). These polypeptides were detectable in autoradiograms prepared from control oocytes at all times of culture, and therefore we have classified them as nondevelopmental polypeptides. The vast majority of the 400 to 500 polypeptides detected were of this type. Even though dbcAMP inhibited gvb in all oocytes examined in our study, and all oocytes examined after release from inhibition had undergone gvb, the synthesis of only three polypeptides was found to be in-
191
hibited reversibly by the analog. Conversely, the synthesis of three polypeptides was irreversibly inhibited by dbcAMP. The three polypeptides whose synthesis was reversibly inhibited by dbcAMP may be of special interest for further analysis, since they could be interpreted to represent specific molecular markers related to the inhibitory influence of cAMP on gvb, and thus to reflect a physiological regulatory activity for the cyclic nucleotide. In contrast, our detection of uninhibited and irreversibly inhibited polypeptides may be the result of nonspecific and pharmacological effects of dbcAMP on the synthesis of polypeptides whose presence or absence is not critical to gvb or polar body extrusion. It is known that several polypeptides begin to be synthesized at various times during oocyte maturation, and continue to be synthesized following fertilization and even during cleavage (Van Blerkom and McGaughey, 1978b). We cannot confirm an earlier report that the pattern of polypeptide synthesis for mouse oocytes cultured continuously in the presence of dbcAMP was "virtually indistinguishable" from that of control oocytes before gvb (Schultz and Wassarman, 1977b). Their results are difficult to interpret since they apparently have displayed the same autoradiogram to demonstrate patterns for both inhibited (i.e., presence of dbcAMP) and maturing (i.e., absence of dbcAMP) oocytes (cf, Figs. 1A and 2B, D of their study). The extremely long culture and radiolabeling period (20 hr) may have resulted in masking of the molecular events which we describe here. In addition, reversal experiments were not reported by them. Our results are not totally inconsistent with the suggestion that cAMP may play a regulatory role in mammalian oocyte maturation (Dekel and Beers, 1978, 1980). However, our results are most consistent with a combination of specific and nonspecific effects by dbcAMP on mouse oocytes. The apparently specific effects (reversible inhibition of polypeptide synthesis) may prove valuable in establishing molecular correlates of gvb, while the nonspecific effects should serve as a serious caution. The developmental patterns of polypeptide synthesis clearly were altered in oocytes exposed to dbcAMP. Since some alterations were irreversible, oo-
(348,000 cpm; 56 oocytes) in the absence of dbcAMP; 0-2 (475,000 cpm; 82 oocytes) in the presence of the dbcAMP, and for reversal (476,000 cpm; 61 oocytes). The arrow in this figure represents a polypeptide which was not detected at 0-2 and 2-4 br but was detected at 4-6 hr both in the presence and absence of dbcAMP and during reversal. (B) Comparison of area B from different autoradiograms. Polypeptides 1 and 2 were detected at 2-4 and 4-6 hr in the absence of dbcAMP, but were not detected at 0-2 hr in the absence of dbcAMP or at any time in the presence of dbcAMP or during reversal. (C) Comparison of area C from different autoradiograms. The arrow refers to a polypeptide detected during 0-2 hr in the presence of dbcAMP, and during 2-4 and 4-6 hr in the absence of dbcAMP and during reversal, but not detected at 2-4 or 4-6 br in the presence of dbcAMP. (D) Comparison of area D from different autoradiograms. Polypeptide 1 was detected at all times (including reversal) in the absence, but not the presence, of dbcAMP. Polypeptide 2 was detected at all times (including reversal) in the absence of cAMP, but was detected only during 4-6 hr in the presence of dbcAMP. Polypeptide 3 was detected only at 2-4 and 4-6 hr in the absence of dbcAMP, but was not detectable in the presence of dbcAMP, or during reversal.
192
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VOLUME83, 1981
peptide synthesis of porcine oocytes during maturation in vitro. Deveh~p. Biol 56, 241-254. MORILL, G. A., SCHATZ,F., KOST/ILOW,A., and POUPKO, J. (1977). Changes in cyclic AMP levels in the amphibian ovarian follicle following progesterone induction of meiotic maturation. D0~erent/at/on 8, 97-104. O'FARRELL, P. H. (1975). High resolution two-dimensional electrophoresis of proteins. J. B/o/, Che~ 204, 4007-4021. RICE, C., and McGAUGHEY,R. W. (1980). The role of the granulosa This work was supported by Grant HD 06532 from the NIH. cell in oocyte maturation. In '"PAth Annual Meeting, Arizona and Nevada Academy of Sciences, Las Vegas, Nevada," Abstr. 100. REFERENCES SCHORDERET-SLATKIN,S., SCHORDERET,M., BOQUET,P., GODEAU,F., and BAULIEU,E. E. (1978). Progesterone-induced meiosis in Xenopus ~ oocytes: A role for cAMP at the "maturation-promoting BRINSTER,R. L. (1971). In vitro culture of the embryo. In "Pathways factor" level. Ce//15, 1269-1275. to Conception, Harold C. Mack Symposium on the Physiology of Reproduction" (A. I. Sherman, ed.) Charles C Thomas, Springfield, SCHULTZ,R. M., and WASSARMA~r P. M. (1977a). Biochemical studies of mammalian oogenesis. Protein synthesis during oocyte growth Ill. and meiotic maturation in the mouse. J. Cell ~ 24, 167-194. CHO, W. K. STERN,S., and BIGGERS,J. D. (1974). Inhibitory effect of dibutyryl cAMP on mouse oocyte maturation in vitro. J. Exp. ZooL SCHULTZ,R. M., and WASSARMAN,P. M. (19r/Tb). Specific changes in the pattern of protein synthesis during meiotic maturation of mam187, 383-386. malian oocytes in vitro. Proc. Nat~ Acad, ~ USA 74, 538-541. DEKEL, N., and BEERS, W. H. (1978). Rat oocyte maturation in vitro: SPEAKER, M. G., and BUTCHER,F. R. (1977). Cyclic nucleotide flucRelief of cyclic AMP inhibition by gonadotropins. Proc. N a t Acad. tuations during steroid induced meiotic maturation of frog oocytes. Sci. USA 75, 4369-4373. Nature (London) 267, 848-849. DEKEL, N., and BEERS, W. H. (1980). Development of the rat oocyte in vitro: Inhibition and induction of maturation in the presence or TSAFRIRI,A., and CHANNING,C. P. (1975). An inhibitory influence of granulosa cells and follicular fluid upon procine oocyte meiosis in absence of cumulus oophorus. Develop. BioL 75, 247-254. vitro. Endocr/no/o~ 96, 922-927. DONAHUE, R. P. (1972). The relationship of oocyte maturation to ovulation in mammals. In "Oogenesis" (J. D. Biggers and A. W. VAN BLERKOM,J. (1978). Methods for the high-resolution analysis of protein synthesis: Applications to studies of early mammalian deSchuetz, eds.). Univ. Park Press, Baltimore. velopment. In "Methods in Mammalian Reproduction" (J. C. Daniel, MALLER, J., and KREBS, E. (1977). Progesterone-stimulated meiotic Jr., ed.). Academic Press, New York. division in Xenopus oocytes. J. BioL Chem. 252, 1712-1718. MALLER,J. WU, M., and GERHART,J. (1977). Changes in protein phos- VANBLERKOM,J., and MCGAUGHEY,R. W. (1978a). Molecular differentiation of the rabbit ovum. I. During oocyte maturation in vivo phorylation accompanying maturation of Xenopus/aev~ oocytes. and in ~itro. Develop. BioL 63, 139-150. De,lop. BioL 58, 295-312. MASUI,Y., and CLARKE,H. J. (1979). Oocyte maturation. Inter~ Re'v. VANBLERKOM,J., and McGAUGHEY,R. W. (1978B). Molecular differentiation of the rabbit ovum. II. During the preimplantation deCytol. 57, 185-282. velopment of in vivo and in vitro matured oocytes. Deve/op. Bled. MCGAUGHEY, R. W. (1977). The culture of pig oocytes in minimal 63, 151-164. medium, and the influence of progesterone and estradiol-17~ on WASSAI~AN, P. M., and I_~rOURNF~U,G. E. (1976). RNA synthesis meiotic maturation. Endocr/~dogy 100, 39-45. in fully-grown mouse oocytes. Nature (London) 361, 73-74. MCGAUGHEY,R. W., and VAN BLERKOM,J. (1977). Patterns of poly-
c y t e s c u l t u r e d i n t h e p r e s e n c e of d b c A M P to m a i n t a i n t h e m a t t h e gv s t a g e c a n n o t be e q u a t e d d e v e l o p m e n t a l l y w i t h u n t r e a t e d i m m a t u r e oocytes. W h e t h e r o r n o t ooc y t e s w h i c h h a v e b e e n a l l o w e d to m a t u r e f o l l o w i n g i n hibition by dbcAMP are competent for fertilization and embryonic development has not been determined.
Patterns of Polypeptide Synthesis in Mouse Oocytes during Germinal Vesicle Breakdown and during Maintenance of the Germinal Vesicle Stage by Dibutyryl cAMP JOEL D. RICHTER 1 A N D R O B E R T W. M C G A U G H E Y Department of Zoology, Arizona State University, Tempe, Arizona 85~81 Received June 19, 1980; accepted in revised farm August 18, 1980 Mouse oocytes undergo spontaneous meiotic maturation when cultured in vitro in the absence, but not in the presence, of dibutyryl cyclic AMP (cAMP). Furthermore, the inhibition by dibutyryl cAMP is reversible. Oocytes synthesize stage-specific polypeptides (i.e., developmental polypeptides) during meiotic maturation. Dibutyryl cAMP affects the synthesis of four of these developmental polypeptides by reversibly inhibiting the synthesis of one polypeptide, and irreversibly inhibiting the synthesis of three other polypeptides. Dibutyryl cAMP also reversibly inhibits the synthesis of at least two specific nondevelopmental polypeptides. INTRODUCTION
Most oocytes of the sexually mature mammal are arrested at late diplotene, a nuclear stage called the germinal vesicle (gv). In response to cyclical release of gonadotropins, some oocytes resume meiosis and undergo the first meiotic division in preparation for ovulation and fertilization. This developmental process, called maturation, will take place in mammalian oocytes which have been isolated from their follicles and maintained in culture (for review, see Donahue, 1972; Masui and Clarke, 1979). Oocyte maturation in vivo and spontaneous maturation in vitro involve not only chromosomal events, but also coordinated changes in synthesis of RNA (Wassarman and Letourneau, 1976) and of protein (McGaughey and Van Blerkom, 1977; Schultz and Wassarman, 1977a; Van Blerkom and McGaughey, 1978a). The regulation of mammalian oocyte maturation has been the subject of many recent studies. Several substances have been reported as potential regulators of oocyte maturation on the basis of their inhibition of spontaneous gv breakdown (gvb) in vitro (Cho et al., 1974; Tsafriri and Channing, 1975; McGaughey, 1977). One of these substances, dibutyryl cyclic adenosine monophosphate (dbcAMP), has been shown to block spontaneous maturation reversibly in oocytes from mice (Cho et al., 1974), rats (Dekel and Beers, 1978), and pigs (Rice and McGaughey, 1980). There is evidence that cyclic nucleotides and protein kinases perform regulatory functions during oocyte 1 Present address: Purdue University, Department of Biological Sciences, West Lafayette, Ind. 47907. 0012-1606/81/050188-05502.00/0 Copyright 9 1981by AcademicPress, Inc. All rights of reproductionin any form reserved.
188
maturation in amphibians (Speaker and Butcher, 1977; Morrill et a/., 1977; Mailer et al., 1977; Schorderet-Slatkin et a/., 1978; Mailer and Krebs, 1977). The mechanism by which dbcAMP acts to block gvb in mammalian oocytes is not known. However, we recently observed that although dbcAMP did not influence measurably the uptake of ~PO42-, its incorporation into phosphoprotein of mouse oocytes was decreased by dbcAMP relative to controls (Richter and McGaughey, in preparation). Since this apparent inhibition of protein phosphorylation may reflect a fundamental role for cAMP in the regulation of maturation in mammals, it has become essential that the influence of the analog on additional developmental parameters be examined. In this study we provide evidence that in the presence of dbcAMP, cultured mouse oocytes exhibit altered patterns of polypeptide synthesis. Our results can be interpreted, in part, to reflect a Potential regulatory function for cAMP in mammalian oocyte maturation. On the other hand, some of the molecular changes were not reversible upon removal of oocytes from the presence of dbcAMP, and therefore may reflect an abnormal, pharmacological influence of the analog on the subsequent development of mouse oocytes. MATERIALS AND METHODS
Oocytes and culture conditions. Oocytes from randomly bred young adult Swiss albino mice were harvested by puncturing ovarian follicles in minimal medium (Brinster, 1971) containing no dbcAMP (control medium) or 100 ~g/ml dbcAMP (Sigma). This dose was chosen as the lowest concentration at which maturation is reversibly inhibited by at least 90% (Cho et al., 1974).
BRIEF NOTES pH Z5
189 pH 43
IO ~= X
b-
.J
0 3E
23-
13FIG. 1. Autoradiogram of a high resolution two-dimensional polypeptide pattern of mouse oocytes. This sample contained 61 oocytes (476,000 cpm of TCA-precipitable radioactivity), which had undergone gvb during culture in control medium for 6 hr, the final 2 hr in the presence of [~S]methionine. Brackets designate areas of the autoradiogram in which the majority of changes in polypeptide patterns were observed. These areas are displayed in detail in the following figure.
Only oocytes which appeared normal under a dissection microscope and which had an intact gv were selected. Oocytes were cultured in 100-~1 drops of either control medium or medium with dbcAMP, in a humidified atmosphere of 5% CO2,5% 02, and 90% N2 at 37~ for 0, 2, or 4 hr, and subsequently for an additional 2 hr in the same medium but containing [35S]methionine (1.06 mCi/ml; spac, 840 Ci/mmole; Amersham). Other oocytes were cultured for a total of 18 hr: 12 hr in the presence of dbcAMP and 6 h r in control medium, the last 2 hr of which were in the radioisotope. Approximately 80% of the oocytes underwent gvb between 2 and 4 hr of culture in control medium. Oocytes which underwent early gvb in the 0- to 2-hr period and those which underwent gvb during the 4- to 6-hr period (with or without an initial 12-hr culture in dbcAMP) were excluded. None of the oocytes from the 2- to 4-hr period was removed. Culture medium with dbcAMP prevented gvb in approximately 95% of the oocytes. Oocytes which did not exhibit an intact gv during any of the culture periods in the presence of dbcAMP were excluded.
Two-dimensional polyacrylamide gel electrophoresis. Labeled oocytes were washed in radioinert medium and placed in 30 ~l of lysis buffer (O'Farrell, 1975) followed by repeated freezing and tha~viag. A 2- ~1 aliquot of the lysate was precipitated in TCA to estimate incorporation of [s5S]methionine. Two-dimensional electrophoresis was performed essentially as described by O'Farrell (1975), except that a linear gradient of 8 to 15% acrylamide was used in the second dimension. Exposures for autoradiograms followed those of Van Blerkom (1978).
Three different atitoradiograms were examined for each culture condition and period. RESULTS AND DISCUSSION
Diburyryl cAMP, but not butyric acid, inhibits mouse oocyte maturation (Cho et al., 1974), and neither butyrate nor cAMP exactly mimic the inhibitory effect of dbcAMP on rat oocytes (Dekel and Beers, 1980). Oocytes cultured for 12 hr in the presence of dbcAMP resume maturation when subsequently cultured in control medium and these reversed oocytes appear to be morphologically normal. We present evidence here that polypeptide synthesis is influenced by dbcAMP but that these molecular alterations are not all reversible. The autoradiograms from high-resolution polyacrylamide gel electrophoresiS were examined within and between experimental groups and were highly consistent for each type of sample. Reproducible changes in patterns of polypeptide synthesis were observed for control oocytes sampled before, during, and after, gvb a n d many of these changes were altered for oocytes in the presence of dbcAMP. A representative au~radiogram (Fig. 1)and portions of autoradiograms (Fig. 2) demonstrate the patterns and changes in patterns which we observed. The brackets in Fig. 1 designate areas in which the majority of changes were identified,and these are displayed in Fig. 2. A distinct progression of changes in pattern occurs during early maturation of control oocytes. Putative developmental polypeptides are shown in Figs. 2A (ar-
190
DEVELOPMENTAL BIOLOGY 0--2
HR
A
2 -- 4
HR
VOLUME83, 1981 4--6
HR
REV.
i 84 9
m
B
"1-
m
C
D
+
FIG. 2. (A) Comparison of area A (Fig. 1) from different autoradiograms. In this and subsequent figures 0-2, 2-4, and 4-6 hr refer to radiolabeling periods following culture in medium without ( - ) or with (+) 100/~g/ml dbcAMP. Reversal (REV) refers to polypeptide patterns during 4-6 hr of culture in control medium after initial culture for 12 hr in medium with dbcAMP. The incorporated radioactivity and numbers of oocytes for the samples in this and subsequent figures were:0-2, (305,000 cpm; 84 oocytes); 2-4 (366,000 cpm; 75 oocytes), 4-6
BRIEF NOTES
row), B (polypeptides 1 and 2), C (arrow), and D (polypeptide 3). In all five cases, these polypeptides were undetectable at 0-2 or 0-4 hr of culture, and were detectable for the first time during or following gvb. These developmental changes are of the same magnitude as those observed in rabbit (Van Blerkom and McGaughey, 1978a) and pig (McGaughey and Van Blerkom, 1977) oocytes during early stages of maturation. However, the synthesis of polypeptides peculiar to the period prior to gvb was not observed, unlike immature oocytes of the pig (McGaughey and Van Blerkom, 1977) and rabbit (Van Blerkom and McGaughey, 1978a). The synthetic patterns of the above developmental polypeptides were influenced in various ways in oocytes in which gvb was inhibited by dbcAMP. One of these polypeptides (Fig. 2A, arrow) exhibited an apparently normal pattern of synthesis (i.e., absent at 0-2hr, present at 2-6 hr) in the absence and presence of dbcAMP. Three (Fig. 2B, Nos. 1 and 2; Fig. 2D No. 3) were not detected in the presence of dbcAMP, although they were synthesized by control oocytes at 2-6 hr of culture. The synthesis of the fifth developmental polypeptide (Fig. 2C, arrow) was inhibited by dbcAMP during 2-6 hr of culture relative to controls; however, surprisingly this polypeptide was detectable at 0-2 hr of culture in the presence of dbcAMP, but not in controls. Reversal of inhibited synthesis for developmental polypeptides was observed in only one case (Fig. 2C, arrow). In the three other cases, polypeptides not detectable in samples of oocytes cultured in dbcAMP also were not detected in autoradiograms from oocytes undergoing reversal of gvb inhibition. The synthesis of two other polypeptides was inhibited reversibly in oocytes cultured in dbcAMP (Fig. 2D, Nos. 1 and 2). These polypeptides were detectable in autoradiograms prepared from control oocytes at all times of culture, and therefore we have classified them as nondevelopmental polypeptides. The vast majority of the 400 to 500 polypeptides detected were of this type. Even though dbcAMP inhibited gvb in all oocytes examined in our study, and all oocytes examined after release from inhibition had undergone gvb, the synthesis of only three polypeptides was found to be in-
191
hibited reversibly by the analog. Conversely, the synthesis of three polypeptides was irreversibly inhibited by dbcAMP. The three polypeptides whose synthesis was reversibly inhibited by dbcAMP may be of special interest for further analysis, since they could be interpreted to represent specific molecular markers related to the inhibitory influence of cAMP on gvb, and thus to reflect a physiological regulatory activity for the cyclic nucleotide. In contrast, our detection of uninhibited and irreversibly inhibited polypeptides may be the result of nonspecific and pharmacological effects of dbcAMP on the synthesis of polypeptides whose presence or absence is not critical to gvb or polar body extrusion. It is known that several polypeptides begin to be synthesized at various times during oocyte maturation, and continue to be synthesized following fertilization and even during cleavage (Van Blerkom and McGaughey, 1978b). We cannot confirm an earlier report that the pattern of polypeptide synthesis for mouse oocytes cultured continuously in the presence of dbcAMP was "virtually indistinguishable" from that of control oocytes before gvb (Schultz and Wassarman, 1977b). Their results are difficult to interpret since they apparently have displayed the same autoradiogram to demonstrate patterns for both inhibited (i.e., presence of dbcAMP) and maturing (i.e., absence of dbcAMP) oocytes (cf, Figs. 1A and 2B, D of their study). The extremely long culture and radiolabeling period (20 hr) may have resulted in masking of the molecular events which we describe here. In addition, reversal experiments were not reported by them. Our results are not totally inconsistent with the suggestion that cAMP may play a regulatory role in mammalian oocyte maturation (Dekel and Beers, 1978, 1980). However, our results are most consistent with a combination of specific and nonspecific effects by dbcAMP on mouse oocytes. The apparently specific effects (reversible inhibition of polypeptide synthesis) may prove valuable in establishing molecular correlates of gvb, while the nonspecific effects should serve as a serious caution. The developmental patterns of polypeptide synthesis clearly were altered in oocytes exposed to dbcAMP. Since some alterations were irreversible, oo-
(348,000 cpm; 56 oocytes) in the absence of dbcAMP; 0-2 (475,000 cpm; 82 oocytes) in the presence of the dbcAMP, and for reversal (476,000 cpm; 61 oocytes). The arrow in this figure represents a polypeptide which was not detected at 0-2 and 2-4 br but was detected at 4-6 hr both in the presence and absence of dbcAMP and during reversal. (B) Comparison of area B from different autoradiograms. Polypeptides 1 and 2 were detected at 2-4 and 4-6 hr in the absence of dbcAMP, but were not detected at 0-2 hr in the absence of dbcAMP or at any time in the presence of dbcAMP or during reversal. (C) Comparison of area C from different autoradiograms. The arrow refers to a polypeptide detected during 0-2 hr in the presence of dbcAMP, and during 2-4 and 4-6 hr in the absence of dbcAMP and during reversal, but not detected at 2-4 or 4-6 br in the presence of dbcAMP. (D) Comparison of area D from different autoradiograms. Polypeptide 1 was detected at all times (including reversal) in the absence, but not the presence, of dbcAMP. Polypeptide 2 was detected at all times (including reversal) in the absence of cAMP, but was detected only during 4-6 hr in the presence of dbcAMP. Polypeptide 3 was detected only at 2-4 and 4-6 hr in the absence of dbcAMP, but was not detectable in the presence of dbcAMP, or during reversal.
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peptide synthesis of porcine oocytes during maturation in vitro. Deveh~p. Biol 56, 241-254. MORILL, G. A., SCHATZ,F., KOST/ILOW,A., and POUPKO, J. (1977). Changes in cyclic AMP levels in the amphibian ovarian follicle following progesterone induction of meiotic maturation. D0~erent/at/on 8, 97-104. O'FARRELL, P. H. (1975). High resolution two-dimensional electrophoresis of proteins. J. B/o/, Che~ 204, 4007-4021. RICE, C., and McGAUGHEY,R. W. (1980). The role of the granulosa This work was supported by Grant HD 06532 from the NIH. cell in oocyte maturation. In '"PAth Annual Meeting, Arizona and Nevada Academy of Sciences, Las Vegas, Nevada," Abstr. 100. REFERENCES SCHORDERET-SLATKIN,S., SCHORDERET,M., BOQUET,P., GODEAU,F., and BAULIEU,E. E. (1978). Progesterone-induced meiosis in Xenopus ~ oocytes: A role for cAMP at the "maturation-promoting BRINSTER,R. L. (1971). In vitro culture of the embryo. In "Pathways factor" level. Ce//15, 1269-1275. to Conception, Harold C. Mack Symposium on the Physiology of Reproduction" (A. I. Sherman, ed.) Charles C Thomas, Springfield, SCHULTZ,R. M., and WASSARMA~r P. M. (1977a). Biochemical studies of mammalian oogenesis. Protein synthesis during oocyte growth Ill. and meiotic maturation in the mouse. J. Cell ~ 24, 167-194. CHO, W. K. STERN,S., and BIGGERS,J. D. (1974). Inhibitory effect of dibutyryl cAMP on mouse oocyte maturation in vitro. J. Exp. ZooL SCHULTZ,R. M., and WASSARMAN,P. M. (19r/Tb). Specific changes in the pattern of protein synthesis during meiotic maturation of mam187, 383-386. malian oocytes in vitro. Proc. Nat~ Acad, ~ USA 74, 538-541. DEKEL, N., and BEERS, W. H. (1978). Rat oocyte maturation in vitro: SPEAKER, M. G., and BUTCHER,F. R. (1977). Cyclic nucleotide flucRelief of cyclic AMP inhibition by gonadotropins. Proc. N a t Acad. tuations during steroid induced meiotic maturation of frog oocytes. Sci. USA 75, 4369-4373. Nature (London) 267, 848-849. DEKEL, N., and BEERS, W. H. (1980). Development of the rat oocyte in vitro: Inhibition and induction of maturation in the presence or TSAFRIRI,A., and CHANNING,C. P. (1975). An inhibitory influence of granulosa cells and follicular fluid upon procine oocyte meiosis in absence of cumulus oophorus. Develop. BioL 75, 247-254. vitro. Endocr/no/o~ 96, 922-927. DONAHUE, R. P. (1972). The relationship of oocyte maturation to ovulation in mammals. In "Oogenesis" (J. D. Biggers and A. W. VAN BLERKOM,J. (1978). Methods for the high-resolution analysis of protein synthesis: Applications to studies of early mammalian deSchuetz, eds.). Univ. Park Press, Baltimore. velopment. In "Methods in Mammalian Reproduction" (J. C. Daniel, MALLER, J., and KREBS, E. (1977). Progesterone-stimulated meiotic Jr., ed.). Academic Press, New York. division in Xenopus oocytes. J. BioL Chem. 252, 1712-1718. MALLER,J. WU, M., and GERHART,J. (1977). Changes in protein phos- VANBLERKOM,J., and MCGAUGHEY,R. W. (1978a). Molecular differentiation of the rabbit ovum. I. During oocyte maturation in vivo phorylation accompanying maturation of Xenopus/aev~ oocytes. and in ~itro. Develop. BioL 63, 139-150. De,lop. BioL 58, 295-312. MASUI,Y., and CLARKE,H. J. (1979). Oocyte maturation. Inter~ Re'v. VANBLERKOM,J., and McGAUGHEY,R. W. (1978B). Molecular differentiation of the rabbit ovum. II. During the preimplantation deCytol. 57, 185-282. velopment of in vivo and in vitro matured oocytes. Deve/op. Bled. MCGAUGHEY, R. W. (1977). The culture of pig oocytes in minimal 63, 151-164. medium, and the influence of progesterone and estradiol-17~ on WASSAI~AN, P. M., and I_~rOURNF~U,G. E. (1976). RNA synthesis meiotic maturation. Endocr/~dogy 100, 39-45. in fully-grown mouse oocytes. Nature (London) 361, 73-74. MCGAUGHEY,R. W., and VAN BLERKOM,J. (1977). Patterns of poly-
c y t e s c u l t u r e d i n t h e p r e s e n c e of d b c A M P to m a i n t a i n t h e m a t t h e gv s t a g e c a n n o t be e q u a t e d d e v e l o p m e n t a l l y w i t h u n t r e a t e d i m m a t u r e oocytes. W h e t h e r o r n o t ooc y t e s w h i c h h a v e b e e n a l l o w e d to m a t u r e f o l l o w i n g i n hibition by dbcAMP are competent for fertilization and embryonic development has not been determined.