Proc. Nati. Acad. Sci. USA Vol. 74, No. 2, pp. 538-541, February 1977 Biochemistry Specific changes in- the pattern of protein synthesis during meiotic maturation of mammalian in vitro (two-dimensional electrophoresis/germinal vesicle) RICHARD M. SCHULTZ AND-PAUL M. WASSARMAN Department of Biological Chemistry and Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, Boston, Massachusetts 02115 Communicated by Don W. Fawcett, November 29,1976

ABSTRACT High resolution two-dimensional electropho- MATERIALS AND METHODS resis has been used to examine the pattern ofprotein synthesis grown oocytes were obtained from adult (8-12 weeks of during meiotic maturation of mouse oocytes in vitro. Fluoro- Fully grams of[IsS methioninelabeled proteins have revealed age), randomly bred, female Swiss albino mice (CD-1, Charles that meiotic progression from dictyate to metaphase II(meiotic River Laboratories) by puncturing isolated ovaries with fine maturation) is accompanied by marked changes in the pattern steel needles under a dissecting microscope (11). Oocytes con- of proteins synthesized by oocytes. Virtually all ofthe changes taining an intact germinal vesicle (GV) and free of cumuluscells observed take place subsequent to the blreakown ofthe oocyte's were harvested with a mouth-operated micropipet and washed germinal vesicle, but are not dependent upon the occurrence in culture medium containing 100 Mg/ml of dibutyryl of other morphological events, such as spindle formation or (12) polar body emission. These changes in protein synthesis do not 3':5'-cyclic AMP (Bt2cAMP) (13, 14). Cell culture was carried take place in oocytes that fail to undergo breakdown ofgerminal out in either plastic dishes (Falcon) or embryological watch vesicles spontaneously or in oocytes arrested at the germinal glasses in 50-250 Ml of medium under paraffin oil at 370 in a vesicle stage by dibutyryl 3':5'-cyclic AMP. These data suggest humidified atmosphere of 5% CO2 air. that mixing of the oocyte's nucleoplasm and cytoplsm may Qocyte proteins labeled with [&aSlmethionine were prepared trigger many of the changes in protein synthesis that accompany for electrophoresis in the following manner: Oocytes cultured meiotic maturation of mouse oocytes in vitro. in the presence of [35SImethionine (200,uCi/ml, New England During the process of , oocytes of many animal species Nuclear) were washed thoroughly and harvested in 2 Ml of undergo meiotic arrest prior to the completion of chromosomal medium. Eight microliters of 0.01 M Tris.HCl, pH 7.4, con- reduction and it is in this state that they undergo tremendous taining 50 jig/ml of RNase was added and the oocytes were growth. The length of time that oocytes remain in this state and frozen and thawed 3 times. The solution was then brought to the nature of the stimulus that reinitiates are species- 9.5 M in urea and 17 Ml of "lysis buffer" (10) was added. A 2-Ml dependent (1-3). aliquot of the solution was assayed by liquid scintillation In mice, nearly all oocytes have arrested at the diplotene counting, and the remainder was subjected to isoelectric fo- stage of of the first meiotic division by cusing and discontinuous sodium dodecyl sulfate (NaDodS04) ("dictyate") gel electrophoresis as described by O'Farrell (10). Gels were 5 days post partum and they remain in dictyate until just prior de- to , a period extending from several weeks to more processed for fluorography according to the procedure than a year. The resumption of meiosis can be mediated by a scribed by Bonner and Laskey (15). hormonal stimulus in vdvo (4) or by the release of oocytes from RESULTS their ovarian follicles into a suitable culture medium (5-8). The oocytes undergo nuclear progression from dictyate to meta- Meiotic maturation takes place spontaneously when oocytes phase II and remain at this stage of meiosis in the oviduct or in from adult mice are released from their ovarian follicles into culture until fertilization or parthenogenetic activation takes a suitable culture medium (5-8). The time sequence of meiotic place. The period of time during which meiosis progresses from maturation in vitro can be approximated as follows: GV dictyate to metaphase II is termed the period of "meiotic breakdown occurs within the first 5 hr, metaphase I is reached maturation." The process of meiotic maturation is characterized in 5-10 hr, and metaphase II is reached in 10-16 hr. Under the by dissolution of the nuclear membrane (germinal vesicle experimental conditions used in this study, approximately 80% breakdown), condensation of diffuse chromatin into distinct of the oocytes placed in culture underwent GV breakdown bivalents, separation of homologous chromosomes and emission within 3 hr and, of these, approximately 70% subsequently of the first polar body, and arrest at metaphase II. Mouse oocytes emitted first polar bodies. matured and fertilized in vitro have developed into viable Mouse oocytes cultured in the presence of Bt2cAMP (100 fetuses after transplantation to the uteri of foster mothers gg/ml) do not undergo GV breakdown or the subsequent (9). morphological events associated with meiotic maturation In this report we describe the results of experiments that used (16-18). Therefore, in order to determine whether changes in high resolution two-dimensional electrophoresis (10) to examine the pattern of protein synthesis occur during meiotic matura- protein synthesis during meiotic maturation of mouse oocytes tion, mouse oocytes were cultured overnight in medium con- in vitro. These experiments show that the pattern of protein taining [35S]methionine in the presence or absence of Bt2cAMP. synthesis changes dramatically as meiosis proceeds and suggest A comparison of fluorograms prepared from oocytes that un- that many of these changes are triggered by mixing of the nu- derwent spontaneous meiotic maturation (Fig. 1B) with fluo- cleoplasm and cytoplasm of the oocytes. rograms prepared from oocytes that were arrested in dictyate by Bt2cAMP (Fig. 1A) reveals a large number of differences. Abbreviations: GV, germinal vesicle; Bt2cAMP, N6,02'-dibutyryl For example, proteins that undergo an increase in their relative adenosine 3':5'-cyclic monophosphate; NaDodSO4, sodium dodecyl rates of synthesis during meiotic maturation are found in re- sulfate. gions D4, D5, F7, F8, G4, and G6 of the fluorograms shown in 538 Downloaded by guest on October 1, 2021 Biochemistry: Schultz and Wassarman Proc. Natl. Acad. Sci. USA 74 (1977) 539

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FIG. 1. High resolution two-dimensional gel electrophoresis of [:35S]methionine-labeled mouse oocyte proteins. The oocytes were collected, labeled with [35S]methionine for 20 hr in the presence (A) or absence (B) of Bt2cAMP (100 jig/ml), and processed for electrophoresis as described in Materials and Methods. Only oocytes with an intact GV were used in (A) and only oocytes that had emitted a polar body were used in (B). The samples, containing 5 X 105 cpm in approximately 20 jl, were focused on 5% polyacrylamide gels containing 2% pH 3-10 Ampholines for 16 hr at 400 V and finally for 1 hr at 800 V. The focusing gels were equilibrated with NaDodSO4 sample buffer (10) for 30 min and subjected to NaDodSO4-polyacrylamide gel electrophoresis at 20 mA per gel at room temperature. The NaDodSO4 separating gel was 12.5 cm long and 1.5 mm thick; the stacking gel was 2.5 cm long. The separating gel was a continuous 9-15% acrylamide gradient (exponential), and the total volume of the gel was 28 mL Approximately 15 oocytes were used per gel. The gels were developed by fluorography (15) for 48 hr; more than 450 spots were detected with this exposure time. Many of the faint spots seen on the fluorogram are not readily seen on the photograph.

Fig. 1. The addition of Bt2cAMP to the culture medium after or puromycin, at the circular bivalent stage by Colcemid, and GV breakdown has occurred does not prevent the changes in at metaphase I by cytochalasin B; the inhibitory effects of these protein synthesis associated with meiotic maturation from drugs are reversible, although the degree of reversibility de- taking place. Furthermore, the pattern of [a5S]methionine- creases over extended periods of culture. In addition to labeled proteins obtained with oocytes cultured continuously Bt2cAMP, Colcemid and cytochalasin B have also been used in the presence of Bt2cAMP is virtually indistinguishable from to determine whether the changes in protein synthesis that that obtained with oocytes that fail to undergo spontaneous accompany meiotic maturation are linked to particular mor- meiotic maturation. phological events, such as spindle formation or polar body Meiotic maturation of mouse oocytes i vitro can be inhibited emission. The results shown in Fig. 2 indicate that these changes by several drugs at specific stages of nuclear progression (18). in protein synthesis do take place when oocytes are cultured For example, oocytes are arrested at the GV stage by Bt2cAMP continuously in the presence of Colcemid (10 ug/ml); similar Downloaded by guest on October 1, 2021 540 Biochemistry: Schultz and Wassarman Proc. Nati. Acad. Sci. USA 74 (1977)

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FIG. 2. High resolution two-dimensional electrophoresis of [-'Slmethionine-labeled proteins from mouse oocytes arrested at various stages of meiosis. Enlargements of selected regions of fluorograms are shown. The oocytes were collected, labeled with [35Slmethionine for 20 hr in plain medium (A and D), in medium containing Bt2cAMP (100,g/ml) (B and E), or in medium containing Colcemid (10 ,ug/ml) (C and F), and were processed for electrophoresis as described in the Materials and Methods. Electrophoresis and fluorography were done as described in the legend to Fig. 1. The arrows (B and Ed) indicate areas of the fluorograms that display characteristic changes in protein synthesis during meiotic maturation. results are obtained with oocytes cultured in the presence of oocyte after stimulation with progesterone also occur in enu- cytochalasin B (5 ,gg/ml). cleated oocytes. Based on the results of their experiments, these In an attempt to correlate changes in the pattern of protein investigators concluded that the mixing of the amphibian synthesis with particular stages of nuclear progression, mouse oocyte's nucleoplasm and cytoplasm is not essential for the oocytes were exposed to [asS]methionine at 5-hr intervals, i.e., successful completion of meiotic maturation or, for that matter, 0-5, 5-10, and 10-15 hr of culture. This protocol permitted an for the activation of the program of protein synthesis that ac- analysis of those proteins synthesized during the period of GV companies nuclear progression. breakdown (0-5 hr), of metaphase I spindle formation (5-10 Unlike the results obtained with amphibian oocytes, our re- hr), and of polar body emission (10-15 hr). The results of these sults indicate that the initiation of those changes in protein experiments are shown in Fig. 3. Although some minor changes synthesis that characterize meiotic maturation of mammalian in protein synthesis can be detected during the first 5 hr of oocytes is dependent upon the mixing of the oocyte's nucleo- culture, the major changes associated with meiotic maturation plasm and cytoplasm. Failure of the mouse oocyte's GV to break take place during the 5-10 hr period after GV breakdown and down, either in medium alone or in the presence of Bt2cAMP, are then accentuated during the 10- to 15-hr period. While prevents all of the changes in protein synthesis that normally there is a decrease in the relative rates of synthesis of many accompany meiotic maturation. On the other hand, once the proteins during the 5- to 15-hr period of meiotic maturation, GV has broken down, the inhibition of subsequent morpho- there are several cases in which the relative rates increase (e.g., logical events, such as spindle formation and/or polar body regions F7, F8, and G6). emission, apparently does not affect the program of changes in protein synthesis that has been set in motion. DISCUSSION The dramatic change in the pattern of protein synthesis in It is well documented that protein synthesis is required for the mouse oocytes after GV breakdown is of interest in view of the successful completion of meiotic maturation of oocytes from reported appearance of a variety of new biochemical activities a variety of species, including echinoderms (19-21), amphibians during meiotic maturation of the amphibian oocyte (29). For (22-24), and mammals (8, 25, 26). Extensive studies done by example, Gurdon and Speight (30) have shown that although Smith and Ecker in vitro (27, 28), using oocytes isolated from the GV, cytoplasm, or a mixture of both from Xenopus laevts Rana pipiens, have shown that there are changes in the nature oocytes are incapable of inducing DNA synthesis in isolated of proteins synthesized during progesterone-induced meiotic somatic nuclei, once GV breakdown has taken place, the egg's maturation. Furthermore, these investigators provided strong cytoplasm can induce DNA synthesis in these nuclei. This ob- evidence to support the contention that, in 'the amphibian servation was extended by Grippo et al. (31), who found that oocyte, the morphological events associated with meiotic a major new DNA polymerase activity appeared during meiotic maturation are under cytoplasmic, not nuclear, control. In order maturation of the amphibian oocyte coincident with GV to test the hypothesis that the program of protein synthesis that breakdown. Although it is not clear whether all of the changes functions during meiotic maturation is directed by templates in protein synthesis that we observe are necessary for the suc- already present in the oocyte's cytoplasm, Smith and Ecker cessful completion of meiotic maturation and/or embryogen- compared the nature of the proteins synthesized in nucleated esis, it seems likely that some of the changes are the result of oocytes with those of enucleated oocytes after exposure to particular events during meiotic maturation itself. For example, progesterone in vitro. They found that those qualitative changes since ribosomal RNA synthesis in the mouse oocytes ceases at in protein synthesis that normally occur in the amphibian the time of GV breakdown (32), it is possible that certain of the Downloaded by guest on October 1, 2021 Biochemistry: Schultz and Wassarman Proc. Natl. Acad. Sd. USA 74 (1977) 541 pH 4.5 < - Electrofocus pH 8.5 RNA synthesis and ribosomal protein synthesis in X. laevis H G F E D C B A oocytes and embryos. A The biochemical basis of meioticmaturation has been studied extensively in amphibians and marine animals. On the other 2 hand, relatively little biochemical information is available concerning meiotic maturation of mammalian oocytes. The 3 experiments described in this report show that such studies on 4 the mammalin oocyte are now technically feasible and suggest that further investigations may reveal additional differences II~ 5 between the biochemistry of oogenesis in mammalian, as compared to nonmammalian, species. 6 .0 S We thank all of the members of our laboratory group for willing 7 assistance and constructive criticism throughout this research. This research was supported by grants awarded to P.M.W. by The National 8 Institute of Child Health and Human Development and The National Science Foundation. R.M.S. is a Postdoctoral Fellow of The Rockefeller B Foundation. 11 -t- w 1. Baker, T. G. (1972) in Reproduction in Mammals, eds. Austin, - I - C. R. & Short, R. V. (Cambridge University Press, London/New York), Vol. 1, pp. 14-45. 2. Schuetz, A. W. (1974) Biol. Reprod. 10, 150-178. 3. Smith, L. D. (1975) in The BRiochemistry of Animal Develop- _wq _a ment, ed. Weber, R. (Academic Press, New York), pp. 1-46. * 4. -Ml _t.x Baker, T. G. (1972) in Reproductive Biology, eds. Balin, H. & .10-q Glasser, S. (Excerpta medica, Amsterdam), pp. 398-437. __.~~~4l, 5. Biggers, J. D., Whittingham, D. G. & Donahue, R. P. (1967) Proc. Natl. Acad. Sci. USA 58,560-567. p 6. Donahue, R. P. (1968) J. Exp. Zool. 169,237-250. 7. Sorensen, R. A. (1973) Am. J. Anat. 136,265-276. 8. Wassarman, P. M. & Letourneau, G. E. (.1976) J. Cell Sci. 20, 549-568. C 9. Cross, P. C. & Brinster, R. L. (1970) Biol. Reprod. 3,298-307. 10. O'Farrell, P. H. (1975) J. Biol. Chem. 250,4007-4021. 11. Rafferty, K. A. (1970) Methods in Experimental Embryology 2 of the Mouse (The Johns Hopkins Press, Baltimore, Md.). 12. Biggers, J. D. (1971) in The Biology of the Blastocyst, ed. 3 Blandau, R. J. (University of Chicago Press, Chicago), pp.

4 319-327. 13. Cho, W. K., Stern, S. & Biggers, J. D. (1974) J. Exp. Zool. 187,

.F, 5 383-386. qw 14. Stern, S. & Wassarman, P. M. (1973) J. Cell Biol. 59, 335a. 6 15. Bonner, W. M. & Laskey, R. A. (1974) Eur. J. Biochem. 46, 83-88. 4 7 16. Wassarman, P. M. (1974) In Vitro 10, 362. 17. Wassarman, P. M. & Turner, P. E. (1976) J. Exp. Zoo!. 196, j8 183-187. 18. Wassarman, P. M., Josefowicz, W. J. & Letourneau, G. E. (1976) FIG. 3. High resolution two-dimensional electrophoresis of J. Cell Sci., in press. [:35S~methionine-labeled proteins from mouse oocytes at various stages 19. Brachet, J. & Steinert, G. (1967) Nature 216, 1314-1315. of meiotic maturation. The oocytes were collected, labeled with 20. Zampetti-Bosseler, F., Huez, G. & Brachet, J. (1973) Exp. Cell [:3S]methionine, and processed as described in the Materials and Res. 78, 383-393. Methods. (A) Oocytes were labeled with [35S]methionine from 0 to 21. Houk, M. S. & Epel, D. (1974) Dev. Biol. 40, 298-310. 5 hr and only those oocytes that had undergone GV breakdown were 22. Brachet, J. (1967) Exp. Cell Res. 48, 233-236. processed. (B) Oocytes were cultured in plain medium for 5 hr and 23. Schuetz, A. W. (1967) J. Exp. Zool. 166,347-354. then only those oocytes that had undergone GV breakdown were 24. Merriam, R. W. (1972) J. Exp. Zool. 180, 421-426. transferred to medium containing [35S]methionine for an additional 25. Stern, S., Rayyis, A. & Kennedy, J. F. (1972) Biol. Reprod. 7, 5-hr culture period before processing. (C) Oocytes were cultured in 341-346. plain medium for 10 hr and then only those oocytes that had under- 26. Schultz, R. M. & Wassarman, P. M. (1977) J. Cell Sci., in gone GV breakdown were transferred to medium containing [35S]- press. methionine for an additional 5-hr culture period before processing; 27. Smith, L. D. & Ecker, R. E. (1969) Dev. Biol. 19,281-309. only oocytes that emitted a polar body were used. The samples, each 28. Ecker, R. E. & Smith, L. D. (1971) Dev. Biol. 24,559-576. containing 750,000 cpm in approximately 20 gl, were subjected to 29. Gurdon, J. B. (1968) Biol. Rev. 43, 233-267. electrophoresis and fluorography as described in the legend in Fig. 30. Gurdon, J. B. & Speight, V. A. (1969) Exp. Cell Res. 55, 253- 1. The gels were developed by fluorography for 36 hr. 256. 31. Grippo, P., Locorotondo, G. & Caruso, A. (1975) FEBS Lett. 51, 137-142. that a decreased relative rate proteins display of synthesis after 32. Wassarman, P. M. & Letourneau, G. E. (1976) Nature 261, GV breakdown are actually ribosomal proteins. The latter 73-74. possibility is enhanced by the results of Hallberg and Brown (33) 33. Hallberg, R. L. & Brown, D. D. (1969) J. Mol. Biol. 46, 393- which suggest that there is coordinated control of ribosomal 411. Downloaded by guest on October 1, 2021