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 oocytes 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
MATERIALS AND METHODS Fully grown oocytes were obtained from adult (8-12 weeks of age), randomly bred, female Swiss albino mice (CD-1, Charles River Laboratories) by puncturing isolated ovaries with fine steel needles under a dissecting microscope (11). Oocytes containing an intact germinal vesicle (GV) and free of cumulus cells were harvested with a mouth-operated micropipet and washed in culture medium (12) containing 100 Mg/ml of dibutyryl 3':5'-cyclic AMP (Bt2cAMP) (13, 14). Cell culture was carried out in either plastic dishes (Falcon) or embryological watch glasses in 50-250 Ml of medium under paraffin oil at 370 in a humidified atmosphere of 5% CO2 air. Qocyte proteins labeled with [&aSlmethionine were prepared for electrophoresis in the following manner: Oocytes cultured in the presence of [35SImethionine (200,uCi/ml, New England Nuclear) were washed thoroughly and harvested in 2 Ml of medium. Eight microliters of 0.01 M Tris.HCl, pH 7.4, containing 50 jig/ml of RNase was added and the oocytes were frozen and thawed 3 times. The solution was then brought to 9.5 M in urea and 17 Ml of "lysis buffer" (10) was added. A 2-Ml aliquot of the solution was assayed by liquid scintillation counting, and the remainder was subjected to isoelectric focusing and discontinuous sodium dodecyl sulfate (NaDodS04) gel electrophoresis as described by O'Farrell (10). Gels were processed for fluorography according to the procedure described by Bonner and Laskey (15).
ABSTRACT High resolution two-dimensional electrophoresis has been used to examine the pattern of protein synthesis during meiotic maturation of mouse oocytes in vitro. Fluorograms of [IsS methioninelabeled oocyte proteins have revealed that meiotic progression from dictyate to metaphase II (meiotic maturation) is accompanied by marked changes in the pattern of proteins synthesized by oocytes. Virtually all of the changes observed take place subsequent to the blreakown of the oocyte's germinal vesicle, but are not dependent upon the occurrence of other morphological events, such as spindle formation or polar body emission. These changes in protein synthesis do not take place in oocytes that fail to undergo breakdown of germinal vesicles spontaneously or in oocytes arrested at the germinal vesicle stage by dibutyryl 3':5'-cyclic AMP. These data suggest that mixing of the oocyte's nucleoplasm and cytoplsm may trigger many of the changes in protein synthesis that accompany meiotic maturation of mouse oocytes in vitro. During the process of oogenesis, oocytes of many animal species undergo meiotic arrest prior to the completion of chromosomal reduction and it is in this state that they undergo tremendous growth. The length of time that oocytes remain in this state and the nature of the stimulus that reinitiates meiosis are species-
dependent (1-3). In mice, nearly all oocytes have arrested at the diplotene ("dictyate") stage of prophase of the first meiotic division by 5 days post partum and they remain in dictyate until just prior to ovulation, a period extending from several weeks to more
than a year. The resumption of meiosis can be mediated by a hormonal stimulus in vdvo (4) or by the release of oocytes from their ovarian follicles into a suitable culture medium (5-8). The oocytes undergo nuclear progression from dictyate to metaphase II and remain at this stage of meiosis in the oviduct or in culture until fertilization or parthenogenetic activation takes place. The period of time during which meiosis progresses from dictyate to metaphase II is termed the period of "meiotic maturation." The process of meiotic maturation is characterized by dissolution of the nuclear membrane (germinal vesicle breakdown), condensation of diffuse chromatin into distinct bivalents, separation of homologous chromosomes and emission of the first polar body, and arrest at metaphase II. Mouse oocytes matured and fertilized in vitro have developed into viable fetuses after transplantation to the uteri of foster mothers
RESULTS Meiotic maturation takes place spontaneously when oocytes from adult mice are released from their ovarian follicles into a suitable culture medium (5-8). The time sequence of meiotic maturation in vitro can be approximated as follows: GV breakdown occurs within the first 5 hr, metaphase I is reached in 5-10 hr, and metaphase II is reached in 10-16 hr. Under the experimental conditions used in this study, approximately 80% of the oocytes placed in culture underwent GV breakdown within 3 hr and, of these, approximately 70% subsequently emitted first polar bodies. Mouse oocytes cultured in the presence of Bt2cAMP (100 gg/ml) do not undergo GV breakdown or the subsequent morphological events associated with meiotic maturation (16-18). Therefore, in order to determine whether changes in the pattern of protein synthesis occur during meiotic maturation, mouse oocytes were cultured overnight in medium containing [35S]methionine in the presence or absence of Bt2cAMP. A comparison of fluorograms prepared from oocytes that underwent spontaneous meiotic maturation (Fig. 1B) with fluorograms prepared from oocytes that were arrested in dictyate by Bt2cAMP (Fig. 1A) reveals a large number of differences. For example, proteins that undergo an increase in their relative rates of synthesis during meiotic maturation are found in regions D4, D5, F7, F8, G4, and G6 of the fluorograms shown in
(9). In this report we describe the results of experiments that used high resolution two-dimensional electrophoresis (10) to examine protein synthesis during meiotic maturation of mouse oocytes in vitro. These experiments show that the pattern of protein synthesis changes dramatically as meiosis proceeds and suggest that many of these changes are triggered by mixing of the nucleoplasm and cytoplasm of the oocytes. Abbreviations: GV, germinal vesicle; Bt2cAMP, N6,02'-dibutyryl adenosine 3':5'-cyclic monophosphate; NaDodSO4, sodium dodecyl sulfate.
538
Biochemistry: Schultz and Wassarman
Proc. Natl. Acad. Sci. USA 74 (1977)
pH 4.5
Vol. 74, No. 2, pp. 538-541, February 1977
Biochemistry
Specific changes in- the pattern of protein synthesis during meiotic maturation of mammalian oocytes 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
MATERIALS AND METHODS Fully grown oocytes were obtained from adult (8-12 weeks of age), randomly bred, female Swiss albino mice (CD-1, Charles River Laboratories) by puncturing isolated ovaries with fine steel needles under a dissecting microscope (11). Oocytes containing an intact germinal vesicle (GV) and free of cumulus cells were harvested with a mouth-operated micropipet and washed in culture medium (12) containing 100 Mg/ml of dibutyryl 3':5'-cyclic AMP (Bt2cAMP) (13, 14). Cell culture was carried out in either plastic dishes (Falcon) or embryological watch glasses in 50-250 Ml of medium under paraffin oil at 370 in a humidified atmosphere of 5% CO2 air. Qocyte proteins labeled with [&aSlmethionine were prepared for electrophoresis in the following manner: Oocytes cultured in the presence of [35SImethionine (200,uCi/ml, New England Nuclear) were washed thoroughly and harvested in 2 Ml of medium. Eight microliters of 0.01 M Tris.HCl, pH 7.4, containing 50 jig/ml of RNase was added and the oocytes were frozen and thawed 3 times. The solution was then brought to 9.5 M in urea and 17 Ml of "lysis buffer" (10) was added. A 2-Ml aliquot of the solution was assayed by liquid scintillation counting, and the remainder was subjected to isoelectric focusing and discontinuous sodium dodecyl sulfate (NaDodS04) gel electrophoresis as described by O'Farrell (10). Gels were processed for fluorography according to the procedure described by Bonner and Laskey (15).
ABSTRACT High resolution two-dimensional electrophoresis has been used to examine the pattern of protein synthesis during meiotic maturation of mouse oocytes in vitro. Fluorograms of [IsS methioninelabeled oocyte proteins have revealed that meiotic progression from dictyate to metaphase II (meiotic maturation) is accompanied by marked changes in the pattern of proteins synthesized by oocytes. Virtually all of the changes observed take place subsequent to the blreakown of the oocyte's germinal vesicle, but are not dependent upon the occurrence of other morphological events, such as spindle formation or polar body emission. These changes in protein synthesis do not take place in oocytes that fail to undergo breakdown of germinal vesicles spontaneously or in oocytes arrested at the germinal vesicle stage by dibutyryl 3':5'-cyclic AMP. These data suggest that mixing of the oocyte's nucleoplasm and cytoplsm may trigger many of the changes in protein synthesis that accompany meiotic maturation of mouse oocytes in vitro. During the process of oogenesis, oocytes of many animal species undergo meiotic arrest prior to the completion of chromosomal reduction and it is in this state that they undergo tremendous growth. The length of time that oocytes remain in this state and the nature of the stimulus that reinitiates meiosis are species-
dependent (1-3). In mice, nearly all oocytes have arrested at the diplotene ("dictyate") stage of prophase of the first meiotic division by 5 days post partum and they remain in dictyate until just prior to ovulation, a period extending from several weeks to more
than a year. The resumption of meiosis can be mediated by a hormonal stimulus in vdvo (4) or by the release of oocytes from their ovarian follicles into a suitable culture medium (5-8). The oocytes undergo nuclear progression from dictyate to metaphase II and remain at this stage of meiosis in the oviduct or in culture until fertilization or parthenogenetic activation takes place. The period of time during which meiosis progresses from dictyate to metaphase II is termed the period of "meiotic maturation." The process of meiotic maturation is characterized by dissolution of the nuclear membrane (germinal vesicle breakdown), condensation of diffuse chromatin into distinct bivalents, separation of homologous chromosomes and emission of the first polar body, and arrest at metaphase II. Mouse oocytes matured and fertilized in vitro have developed into viable fetuses after transplantation to the uteri of foster mothers
RESULTS Meiotic maturation takes place spontaneously when oocytes from adult mice are released from their ovarian follicles into a suitable culture medium (5-8). The time sequence of meiotic maturation in vitro can be approximated as follows: GV breakdown occurs within the first 5 hr, metaphase I is reached in 5-10 hr, and metaphase II is reached in 10-16 hr. Under the experimental conditions used in this study, approximately 80% of the oocytes placed in culture underwent GV breakdown within 3 hr and, of these, approximately 70% subsequently emitted first polar bodies. Mouse oocytes cultured in the presence of Bt2cAMP (100 gg/ml) do not undergo GV breakdown or the subsequent morphological events associated with meiotic maturation (16-18). Therefore, in order to determine whether changes in the pattern of protein synthesis occur during meiotic maturation, mouse oocytes were cultured overnight in medium containing [35S]methionine in the presence or absence of Bt2cAMP. A comparison of fluorograms prepared from oocytes that underwent spontaneous meiotic maturation (Fig. 1B) with fluorograms prepared from oocytes that were arrested in dictyate by Bt2cAMP (Fig. 1A) reveals a large number of differences. For example, proteins that undergo an increase in their relative rates of synthesis during meiotic maturation are found in regions D4, D5, F7, F8, G4, and G6 of the fluorograms shown in
(9). In this report we describe the results of experiments that used high resolution two-dimensional electrophoresis (10) to examine protein synthesis during meiotic maturation of mouse oocytes in vitro. These experiments show that the pattern of protein synthesis changes dramatically as meiosis proceeds and suggest that many of these changes are triggered by mixing of the nucleoplasm and cytoplasm of the oocytes. Abbreviations: GV, germinal vesicle; Bt2cAMP, N6,02'-dibutyryl adenosine 3':5'-cyclic monophosphate; NaDodSO4, sodium dodecyl sulfate.
538
Biochemistry: Schultz and Wassarman
Proc. Natl. Acad. Sci. USA 74 (1977)
pH 4.5
