DEVELOPMENTAL
BIOLOGY
Qualitative
44, 148-157 (19%)
Patterns of Protein Synthesis
in the Preimplantation
Mouse Embryo I. Normal JONATHAN Department
of Molecular,
Cellular
VAN
Pregnancy
BLERKOM’
AND GARY
and Developmental Biology, 80302 Accepted January
0. BROCKWAY
University
of Colorado,
Boulder,
Colorado
10, 1975
Qualitative patterns of protein synthesis in preimplantation mouse embryos were examined by SDS-polyacrylamide-gel electrophoresis followed by autoradiography. The results demonstrate that the qualitative pattern of protein synthesis in newly fertilized eggs (day 1) is very similar to the protein pattern obtained from ovulated, unfertilized eggs. By late day 1 or early day 2, most of these “maternal” proteins are no longer being synthesized by the embryo, and many new autoradiographic bands are apparent. The most intriguing aspect of this study is the observation that all major changes in the qualitative pattern of protein synthesis take place between fertilization and the four- to eight-cell stage (day 3). From early day 3 onward, the qualitative pattern of protein synthesis remains essentially unchanged. Many of the major autoradiographic bands observed in mouse embryos from the four- to eight-cell stage and onward are also observed in protein patterns obtained from blastocyst-stage rabbit embryos. The changing patterns of protein synthesis revealed in this study occur before any gross differentiation of the embryos is evident (delineation of the inner cell mass and trophoblast) and before a marked increase in the relative rate of incorporation of L- [Y!i]methionine takes place. However, the qualitative changes in the pattern of protein synthesis do coincide with a period of extensive fine structural differentiation. INTRODUCTION
The preimplantation period of development of the mammalian embryo has been most extensively studied in the mouse. Studies describing changes in fine structure (Hillman and Tasca, 1969; McReynolds and Hadek, 1972), enzymatic activities (Biggers and Stern, 1973; Brinster, 1973; Graham, 1973a), metabolism and metabolic pathways (Biggers and Stern, 1973; Whitten, 1971), and macromolecular synthesis (Brinster, 1973; Graham, 1973a,b) have yielded a wealth of detailed information concerning early mouse embryogenesis. Results obtained from many of these studies are relatively unequivocal insofar as the reported observations are ‘Present address: B. F. Stolinsky Laboratory, Department of Pediatrics, University of Colorado Medical Center, Denver, CO 80220 and to whom reprint requests should be addressed. Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
148
generally in agreement. By contrast, results obtained by different investigators who have examined protein synthetic activities of preimplantation mouse embryos are in conflict. On the one hand, studies by Weitlauf and his collaborators (see Weitlauf (1974) for a summary) have been interpreted as indicating that no embryonic protein synthesis takes place in uiuo prior to blastocyst formation. On the other hand, experiments in which preimplantation mouse embryos have been exposed to radiolabeled amino acids in vitro suggest that protein synthesis occurs from the two-cell stage onward (Mintz, 1964; Brinster, 1973). Recently, Weitlauf (1974) attempted to resolve this discrepancy by suggesting that incorporation of labeled amino acids by cleaving embryos in vitro may primarily reflect “the turnover of existing protein and the synthesis of a small amount of a
VAN BLERKOM
AND BROCKWAY
Protein
Synthesis
in Preimplantation
Mouse Embryos
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specific protein involved in cleavage,” and microscopically to ensure that fertilization that the “increased incorporation shown had occurred and to determine the stage of after the morula and blastocyst stages in development and then placed into the vitro or at the blastocyst stage in uiuo” radioactive-labeling medium described bemay reflect the “beginning of a marked low. Eggs and follicle cells were obtained increase in the synthesis and accumulation from superovulated HS mice according to of new protein.” the procedure of Gates (1971). Follicle In the present communication, we pre- cells were removed from either eggs or sent results obtained from an analysis of early day 1 embryos by treatment with qualitative patterns of protein synthesis in hyaluronidase (100 international units follicle cells, ovulated, unfertilized eggs, (I.U.) per ml of culture medium, Worthingand cleavage- and blastocyst-stage em- ton Biochemical Corp.) for several minbryos. Because preimplantation mouse em- utes. Isotopic labeling of embryonic proteins. bryos do not incorporate labeled amino The culture medium used in these studies acids in uiuo until the blastocyst stage (Weitlauf, 1974), we have examined rates was that of Whitten (1971) with the follow(1) No protein suppleof incorporation and protein synthetic pat- ing modifications: terns of eggs and embryos labeled in vitro ment (such as bovine serum albumin) was with L- [YSlmethionine for short periods of present and (2) the organic buffer HEPES, time. Protein synthetic patterns were ob- N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Calbiochem) was added to tained by sodium dodecyl sulfate 20 mA4 at pH 7.4 (Van Blerkom and (SDS)-polyacrylamide-gel electrophoresis Manes, 1974). Radioactive culture medium in exponential gradient slab-gels followed was prepared by adding 0.475 ml of Whitby autoradiography (Van Blerkom and ten’s medium to 0.025 ml of L-[35S]methiManes, 1974). Our results indicate that not onine (New England Nuclear; specific aconly is the preimplantation period charactivity, 150 Ci/mmole) such that the final terized by a changing and complex pattern concentration of label in the medium was of protein synthesis but also that all major 200 pCi/ml. Radioactive methionine supqualitative changes in the pattern of proplied by the manufacturer is in water at 100 tein synthesis occur prior to approximately pCi/ml and contains 2-mercaptoethanol as the four- to eight-cell stage. a reducing agent. In a typical experiment. the 2-mercaptoethanol was removed, and MATERIALS AND METHODS the volume of the stock solution of labeled Embryo collection. Embryos were ob- methionine was reduced from 0.1 ml to 0.025 ml by lyophilization. tained from sexually mature, randomly Embryos were collected from the reprobred HS mice (Heterogeneous Stock deductive tract at three daily intervals, early, scribed by McClearn et al., 1970) following spontaneous ovulation. Females were mid, and late, during the entire preimplanplaced with males at 6 PM and checked for tation period. For eggs and cleaving embryos, culture in radioactive medium vaginal plugs a 8 AM the following morning. lasted 2 hr, according to the following Embryonic age is given as days following fertilization, with day 1 being the first day schedule: 8-10 AM (early), 2-4 PM (mid), and 9-11 PM (late). Blastocyst-stage emthe vaginal plug was detected. Animals were sacrificed by cervical dislocation, and bryos were exposed to label for 1 hr at the the oviducts and uteri were isolated and times indicated above. Between 25 and 40 embryos were labeled at one time for each either cut into small pieces in culture stage examined, and the labeling of each medium (oviducts) or flushed with culture embryonic stage was repeated at least six medium (uteri). Embryos were examined
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DEVELOPMENTALBIOLOGY
times. After exposure to label, all embryos appeared grossly normal. Ovulated, unfertilized eggs (with follicle cells removed) were labeled for 2 hr. Preparation of embryos for determinaand electrophoresis. tion of incorporation Following exposure to L- [35S]methionine, embryos were washed in several changes of warm, unlabeled medium (10 ml/wash). After the sequential washes, embryos or eggs were transferred in approximately 10 ~1 of medium to l-ml tubes containing 30 ~1 of lysis buffer (Van Blerkom and Manes, 1974). Both SDS and 2-mercaptoethanol are included in this buffer; to ensure complete lysis of the embryos, the samples were heated in boiling water for 1 min. The embryonic or egg samples were either used for electrophoresis or for determination of incorporation. Activities of samples (cpml ~1) were determined by precipitating the radiolabeled protein mixture (still in approximately 30 ~1 of lysis buffer) in 10% trichloroacetic acid (TCA), heating the mixture at 90°C for 10 min to hydrolyze any charged transfer ribonucleic acid, and then cooling the mixture at 5°C. The chilled samples were passed through Millipore filters with subsequent washes of cold 5% TCA and given a final rinse of cold 95% ethanol. The Millipore filters were dried, and retained radioactivity was measured under a toluene-PPO-POPOP scintillation mixture in a Beckman LS-250 counter. The relative rates of incorporation were determined from the activities of the samples, the number of embryos or eggs in each sample, and the duration of labeling. Follicle cells were labeled and prepared in the same manner as the embryos and eggs with the exception that the duration of labeling was 1 hr. Preparation and running of exponential acrylamide slab-gels. Exponential gradient gels containing 8-15% acrylamide were prepared as previously described Van Blerkom and Manes, 1974). Approximately 40,000 cpm was applied to each of the 15 separate wells on a slab. A typical exposure to X-ray
VOLUME 44, 1975
film (Kodak No-Screen medical X-ray film, type NS2T) lasted 14 days. Molecular weights of embryonic proteins were estimated using both radioactive prereplicative bacteriophage T4 proteins and several other proteins such as the B, B’ subunits of E. coli RNA polymerase, RNase, lysozyme, myoglobin, bovine serum albumin, and chymotrypsinogen A (Van Blerkom and Manes, 1974). RESULTS
Quantitatiue
Aspects of Protein Synthesis
Incorporation of L- [YS]methionine into TCA-insoluble material by preimplantation mouse embryos measured at three daily intervals is shown in Fig. 1. The results are expressed as cpm per embryo per hr and represent values obtained from embryos exposed to label in vitro for a period between 1 and 2 hours (for each point). From the relative rates of incorporation, it is evident that, on a per embryo basis, no significant change occurs from early day 1 until some time between late day 3 and early day 4. In fact, there is a slight but reproducible decrease in incorporation between early day 1 and mid-to-late day 1. From late day 1 until approximately late day 3, mouse embryos incorporate about 500 cpmlhrlembryo when exposed to label of the same specific activity (150 Ci/mmole). An increase in incorporation is initially observed between late day 3 and early day 4 and continues to rise rapidly until implantation on mid day 5. The relative rate of incorporation on mid day 4 is approximately sixfold greater than previously and increases to 13-fold late on day 4. By mid day 5, embryos incorporate label at approximately 25 times the rate observed during cleavage. These values reflect only relative rates of incorporation, because measurements of uptake and endogenous pools of methionine were not undertaken. Qualitative Aspects of Protein Synthesis Qualitative changes in the pattern of embryonic protein synthesis throughout
VAN BLERKOM
AND BROCKWAY
SO
Protein Synthesis in Preimplontation Mouse Embryos
151
l \ *-.-o-o-.-.-.
ii 0
“““““““J El MI MI E2 Y2 M2 E3 M3 MS E4 M4 Y4 E5 &,LANTATION MI LI Y2 L2 MS L3 Y4 L4 M5 DAYS
FOLLOWING
FERflLlZATlON
1. The relative rates of incorporation of L- [%]methionine into TCA-insoluble material throughout the preimplantation period. Incorporation values were obtained at 3 daily intervals, early (E), mid (M), and late FIG.
ad. the entire preimplantation period are shown in Figs. 2 and 3. The electrophoretic separation and resolution of protein bands in this type of gel system (approximately 110 autoradiographic bands) is such that changes in the pattern of protein synthesis from one day to another are largely self-evident. As a consequence and in order not to increase the complexity of the autoradiograms, the actual identification of bands has been kept to a minimum. However, from a careful examination of each column, it is possible to obtain a general appreciation of the changing qualitative pattern of protein synthesis during early mouse embryogenesis. The qualitative pattern of protein synthesis in follicle cells, ovulated, unfertilized eggs, and early cleaving embryos is presented in Fig. 2. It is clear from this figure that follicle cells and unfertilized eggs are engaged in the synthesis of a complex pattern of proteins that range across the entire gel (and that have molecular weights ranging from approximately 8,000 at the bottom of the gel, to 250,000 at the top). It is also evident that proteins
synthesized by follicle cells are, for the most part, qualitatively different from proteins synthesized by unfertilized eggs. On the other hand, a significant degree of overlap exists between proteins synthesized by unfertilized eggs and day-l embryos. This overlap is especially evident in regions a-d in Fig. 2. It is also clear that day-l embryos synthesize proteins that are not being produced either by unfertilized eggs or follicle cells. The most outstanding examples of these proteins are located at approximately 35,000 daltons (arrows e) . What is most remarkable about these proteins is that not only do they represent a considerable portion of the total protein synthetic activity of day 1 embryos but also that the duration of their synthesis is less than 24 hr. Two characteristic features of the protein synthetic patterns of day-2 embryos are the appearance of a new set of autoradiographic bands and, of probably greater developmental significance, the disappearance of most bands observed in ovulated, unfertilized eggs and day-l embryos. Major new autoradiographic bands are evident in
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FOLLICLE CELLS
DEVELOPMENTALBIOLOGY VOLUME44, 1975
UNFERT;‘G;ED DAYS
El
LI
FOLLOWING
-
APPROXIMATE
I
E2
L2
FERTILIZATION l-2
NUMBER
2
2-4
OF CELLS
FPIG.2. The ! qualitative pattern (autoradiograph) of protein synthesis in follicle cells, ovulated, unfertiliz :ed eggs5 and early (El, E2) and late (Ll, L2) l- and e-day embryos. The approximate number of cells per embl :yo was determine sd after labeling. Approximate molecular weight values x lOA are given on the right.
E3L3
E4 M4
M4 L4
E5 M5
RABBIT BLASTOCYST
DAYS FOLLOWING FERTILIZATION 4-816
30
APPROXIMATE FIG. 3. The qualitative
60 NUMBER
100
-
OF CELLS
pattern (autoradiograph) of protein synthesis obtained from day-3, day-4 and day-5 embryos. No differences were observed when embryos labeled early (E), mid (M) and late (L) in each stage were compared. One qualitative difference in the pattern of protein synthesis in older embryos is evident on early-to-mid day 5; two bands located at approximately 12,000 daltons are not observed previously (arrow, E5-M5). The qualitative pattern of protein synthesis in 4.5-day-old rabbit embryo (blastocyst) is demonstrated in the column on the right, and bands common to mouse and rabbit embryos are indicated by arrows. The approximate number of cells per embryo was determined after labeling. Approximate molecular weight values x 10m3 are given at the right. 153
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DEVELOPMENTALBIOLOGY
regions f-j in Fig. 2. A comparison of early-to-mid day-2 patterns with late day-2 patterns reveals several qualitative differences. For example, several new proteins synthesized late on day 2 are observed in regions k-n. The final observed change in the qualitative pattern of protein synthesis during the preimplantation period occurs at some point between the four- and eight-cell stage (i.e., between early and mid day 3). Protein synthetic patterns obtained from day-3 and older mouse embryos are shown in Fig. 3. From approximately mid day 3 and for the remainder of the preimplantation period, the pattern of protein synthesis as revealed by this electrophoretic technique remains remarkably uniform and constant. Proteins synthesized by day-3 and older embryos are for the most part qualitatively different from proteins synthesized by earlier embryos. This qualitative difference is evident if Fig. 2 and 3 are closely compared. Of special interest in day-3 protein synthetic patterns are autoradiographic bands in the relatively low molecular weight region of the gel (S,OOO-15,000). Typically, these particular bands appear for the first time during the preimplantation period very early on day 3. The qualitative pattern of protein synthesis in a 4.5-day-old rabbit embryo is presented in the last column in Fig. 3. One feature that is quite clear from a comparison of mouse and rabbit protein synthetic patterns is that most of the major proteins produced by mouse embryos from day 3 onward are also synthesized by rabbit blastocysts. These particular bands are denoted by arrows in Fig. 3. Although the identity of the proteins composing these bands is unknown, preliminary studies indicate that in the mouse (as in rabbit blastocysts, Van Blerkom and Manes, 1974), actin is the major protein synthesized by embryos from day 3 onward (arrow a) and also by follicle cells. One additional protein, located between 50,000 and 55,000 daltons, has been tentatively identified as
VOLUME 44, 1975
tubulin (arrow t) (Van Blerkom, lished observations).
unpub-
DISCUSSION
Quantitative studies of the incorporation into TCA-insoluble of L- [35S]methionine material by preimplantation mouse embryos labeled in vitro indicate that no significant change occurs until some point between late day 3 and early day 4. From early day 4 until implantation on mid day 5, the rate of incorporation rises rather dramatically. The absolute rate of embryonic protein synthesis is a function of cell number, uptake, endogenous pool size, turnover rate, and the amino acid(s) used as a radioactive tracer (Epstein and Smith, 1973; Borland and Tasca, 1974). The relative rate of incorporation obtained in the present study should be interpreted with care as only incorporation into TCAinsoluble material was measured, and these values may not reflect the actual rate of embryonic protein synthesis. However, the rates of incorporation presented here are in general agreement with previous studies which indicated that no marked increase in incorporation occurs prior to approximately day 3 and that, after day 3, the rate of incorporation of radiolabeled amino acids into protein rises rapidly (Monesi and Salfi, 1967; Tasca and Hillman, 1970; Brinster, 1971, 1973; Epstein and Smith, 1973). Several cautions on the interpretation of autoradiographic patterns of embryonic protein synthesis were discussed in a previous paper (Van Blerkom and Manes, 1974). However, a few major points are worth restating here. With this type of gel system, approximately 110 autoradiographic bands can be resolved. For the most part, each band represents a heterogeneous collection of proteins that have the same approximate molecular weight. Thus, proteins separated by electrophoresis and visualized by autoradiography are those that would be either individually or collectively present in relatively high concentrations.
VAN BLERKOM
AND BROCKWAY
Protein Synthesis in Preimplantation
With these cautions on interpretation in mind, several qualitative aspects of embryonic protein synthesis are evident from an analysis of the autoradiographs. Most proteins synthesized by day-l embryos (and resolved in this gel system) also appear to be synthesized by ovulated, unfertilized eggs but not by follicle cells. By day 2, most of these “early” proteins are no longer being produced or are synthesized at a greatly reduced level (i.e., by the two-cell stage). If proteins synthesized by ovulated eggs are considered to be “maternal” proteins, then it is quite evident that the newly fertilized egg continues to produce primarily maternal proteins. That newly fertilized eggs are not synthesizing exclusively maternal proteins is clear from an examination of day-l protein synthetic patterns. Several proteins synthesized by day-l embryos are not observed in patterns obtained from unfertilized eggs. The major examples of these proteins are located at approximately 35,000 daltons and represent a significant percent of the total protein synthetic activity of the early embryo. Protein synthetic patterns of day-2 embryos reveal several interesting features of this stage of development. First, most proteins synthesized on day 1 (and by eggs) are no longer being produced on day 2. Most of the day-2 proteins resolved in this system are synthesized for approximately 24 hr or less. Secondly, qualitative differences are evident when early and late day-2 protein synthetic patterns are compared. Although these differences are reproducible, their developmental significance is unknown. The qualitative pattern of protein synthesis observed in embryos from early to mid day 3 (four- to eight-cell stage) and from that point onward is remarkable for the following reasons: (1) The qualitative patterns are significantly different from what was observed previously, and (2) no further qualitative changes in the pattern of protein synthesis take place for the
Mouse Embryos
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remainder of the preimplantation period. Many “new” proteins are synthesized by day-3 embryos, and, of these, several are of particular interest because they represent the major species of proteins synthesized by embryos from about the four-cell stage onward. A comparison of mouse protein patterns in day-3 and older embryos with 4.5-day-old rabbit embryos indicates a striking degree of similarity. The bands that contain the major species of proteins in day-3 and older mouse embryos are also the major bands of proteins in rabbit blastocysts. The major protein synthesized by mouse and rabbit embryos (and follicle cells) appears to be actin (Van Blerkom and Manes, 1974). Although the identity of the other major proteins common to mouse and rabbit embryos is unknown, one possibility is that they represent proteins involved in the maintenance of the structural integrity of the embryo and are required for the formation of the blastocyst (Van Blerkom and Manes, 1974). At present, it is quite difficult to interpret the developmental significance of the qualitative changes in the pattern of protein synthesis. This is true not only because the proteins that compose the autoradiographic bands are unidentified but also because little is known relevant to their origin (maternal vs embryonic mRNA). There are, however, several possibilities that may be helpful in attempting to understand what these patterns may mean in the course of early mouse embryogenesis. Numerous changes in enzyme constitution and activity of preimplantation mouse embryos have been studied in detail (Biggers and Stern, 1973; Brinster, 1973). Although it is doubtful whether most enzymes would be present in the embryo in amounts sufficient to be resolved individually in a study of whole embryos, enzymatic changes cannot be excluded from consideration. Alternately, the synthesis of many proteins may depend upon the participation of the paternal genome. However, studies with partheno-
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genie cleavage and blastocyst-stage mouse embryos demonstrate that the qualitative pattern of protein synthesis in these embryos is essentially identical to patterns obtained from normally fertilized eggs at the same stage of development (Van Blerkom and Runner, unpublished observations) . Although the qualitative pattern of protein synthesis remains invariant during the period of gross morphologic differentiation (i.e., blastocyst formation and the elaboration of the inner cell mass and trophoblast), many of the observed changes occur during a period of rapid fine structural differentiation. Comprehensive studies of the fine structural differentiation of the preimplantation mouse embryo (Calarco and Brown, 1969; Hillman and Tasca, 1969) have demonstrated that the development and differentiation of major organelle systems are initiated at fertilization and are essentially complete by the eight- to twelve-cell stage. Major fine structural changes that occur during this period include the following: (1) An increase in the number of cytoplasmic vesicles, crystalloid bodies and ribosomes, (2) differentiation and activation of mitochondria and nucleoli, and (3) elaboration of the rough-surfaced endoplasmic reticulum and formation of junctional complexes. Possibly, the changing patterns of protein synthesis observed during early mouse development are correlated with fine structural differentiation known to occur during this period. A similar correlation between fine structural differentiation and changing qualitative patterns of protein synthesis has been demonstrated during preimplantation development in the rabbit (Van Blerkom et al., 1973; Van Blerkom and Manes, 1974). In the rabbit, as in the mouse, the major qualitative changes in the pattern of protein synthesis occur during cleavage when most of the major fine structural alterations are initiated and, in most cases, completed. This observation is rather surprising in light of the incorporation results
VOLUME 44, 1975
presented earlier and indicates that the increase in the relative rate of incorporation of radiolabeled amino acid(s) occurs both after the fine structural differentiation of the embryo is largely completed and after most changes in the qualitative pattern of protein synthesis have occurred. An additional point to be considered concerns the nature of differential gene expression in preimplantation development. As is evident from a detailed examination of both mouse and rabbit protein synthetic patterns (Van Blerkom and Manes, 1974), many proteins are synthesized for only a relatively short period of time, and, of these, some are produced in significant amounts. It has become increasingly apparent from these studies that early mammalian development includes not only the progressive “turning on” of genetic information, but also, and perhaps of equal importance, a relatively rapid “turning off” of this information and halt in the synthesis of specific proteins. Perhaps both the turning on of genes and the scheduled shut-off of their expression represent functions that are an integral part of an overall autonomous developmental program contained within the genome. Finally, as mentioned earlier, results obtained from quantitative studies of early embryonic protein synthesis in vivo and in vitro are in conflict (Weitlauf, 1974). Although our results do not clarify this discrepancy, they do demonstrate that, in a qualitative sense, the entire preimplantation period is characterized by a complex and changing (at least in the early phases) pattern of protein synthesis. Experiments currently in progress should establish whether these autoradiographic patterns, obtained from embryos labeled in vitro for relatively brief periods of time, reflect actual in vivo protein synthetic activity. Addendum. After this paper was submitted, a study by C. J. Epstein and S. A. Smith of qualitative patterns of protein synthesis by preimplantation mouse embryos appeared in Developmental Biology
VAN BLERKOM AND BROCKWAY
Protein
Sj nthesis in Preimplantation
(Develop. Biol. 40, 233-244 (1974)). Results obtained in that study are in general agreement with the results presented in this communication. We express our appreciation to Dr. Cole Manes for his continued support and helpful criticisms. Funds for this work were made available from a grant to Dr. Cole Manes (Grant No. HD-04274 from the National Institutes of Health, United States Public Health Service). REFERENCES BIGGERS, J. D., and STERN, S. (1973). Metabolism of the preimplantation mammalian embryo. In “Advances in Reproductive Physiology” (M. W. H. Bishop, ed.) Vol. 6, pp. 1-59. Paul Elek Scientific Books, London. BORLAND, R. M., and TASCA, R. J. (1974). Activation of a Na+-dependent amino acid transport system in preimplantation mouse embryos. Deuelop. Biol. 36, 1699182. BRINSTER, R. L. (1971). Uptake and incorporation of amino acids by the preimplantation mouse embryo. J. Reprod. Fert. 27, 329-338. BRINSTER, R. L. (1973). Protein synthesis and enzyme constitution of the preimplantation mammalian embryo. In “The Regulation of Mammalian Reproduction” (S. Segal, R. Crozier, P. A. Corfman, and P. G. Condliffe, eds.). pp. 3022334. C. C Thomas, Springfield, IL. CALARCO, P. G., and BROWN, E. H. (1969). An ultrastructural and cytological study of preimplantation development of the mouse. J. Exp. Zool. 171, 253-283. EPSTEIN, C. J., and SMITH, S. (1973). Amino acid uptake and protein synthesis in preimplantation mouse embryos. Deuelop. Biol. 33, 171-184. GATES, A. H. (1971). Maximizing yield and developmental uniformity of eggs. In “Methods in Mammalian Embryology” (J. C. Daniel, ed.), pp. 64-75. W. H. Freeman, San Francisco. GRAHAM, C. F. (1973a). The necessary conditions for gene expression during early mammalian development. In “Genetic Mechanisms of Development” (31st Symppsium, The Society for Developmental
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Biology) (F. H. Ruddle, ed.), pp. 202-224. Academic Press, New York. GRAHAM, C. F. (1973b). Nucleic acid metabolism during early mammalian development. In “Regulation of Mammalian Reproduction” (S. Sepal, R. Crozier, P. A. Corfman, and P. G. Condliffe, eds.), pp. 286-301. C. C Thomas, Springfield, IL. HILLMAN, N., and TASCA, R. J. (1969). Ultrastructural and autoradiographic studies of mouse cleavage stages. Amer. J. Anat. 126, 151-174. MCCLEARN, G. E., WILSON, J. R., and MEREDITH, W. (1970). The use of isogenic and heterogenic mouse stocks in behavioral research. In “Contributions to Behavior-Genetic Analysis-The Mouse as a Prototype” (G. Lindzey and D. D. Thiessen, eds.), pp. 3-22. Appleton-Century-Crofts. MCREYNOLDS, H. D., and HADEK, R. (1972). A comparison of the fine structure of late mouse blastocysts developed in uiuo and in uitro. J. Exp. Zool. 182, 95-118. MINTZ, B. (1964). Synthetic processes and early development in the mammalian egg. J. Exp. Zool. 157, 85-100. MONESI, V., and SALFI, V. (1967). Macromolecular synthesis during early development in the mouse embryo. Exp. Cell Res. 46, 632-635. TASCA, R. J., and HILLMAN, N. (1970). Effects of actinomycin D and cycloheximide on RNA and protein synthesis in cleavage stage mouse embryos. Nature (London) 225, 1022-1025. VAN BLERKOM, J., ~~~MANEs, C. (1974). Development of preimplantation rabbit embryos in uiuo and in uitro. II. A comparison of qualitative aspects of protein synthesis. Develop. Biol. 40, 40-51. VAN BLERKOM, J., MANES, C., and DANIEL, J. C., JR. (1973). Development of preimplantation rabbit embryos in uiuo and in uitro. I. An ultrastructural comparison. Deuelop. Biol. 35, 262-282. WEITLAUF, H. M. (1974). Metabolic changes in the blastocysts of mice and rats during delayed implantation. J. Reprod. Fert. 39, 213-224. WHITTEN, W. K. (1971). Nutrient requirements of the culture of preimplantation embryos in uitro. In “Advances in the Biosciences” (G. Rasp& ed.), Vol. 6, pp. 129-141. Pergamon Press, London.
BIOLOGY
Qualitative
44, 148-157 (19%)
Patterns of Protein Synthesis
in the Preimplantation
Mouse Embryo I. Normal JONATHAN Department
of Molecular,
Cellular
VAN
Pregnancy
BLERKOM’
AND GARY
and Developmental Biology, 80302 Accepted January
0. BROCKWAY
University
of Colorado,
Boulder,
Colorado
10, 1975
Qualitative patterns of protein synthesis in preimplantation mouse embryos were examined by SDS-polyacrylamide-gel electrophoresis followed by autoradiography. The results demonstrate that the qualitative pattern of protein synthesis in newly fertilized eggs (day 1) is very similar to the protein pattern obtained from ovulated, unfertilized eggs. By late day 1 or early day 2, most of these “maternal” proteins are no longer being synthesized by the embryo, and many new autoradiographic bands are apparent. The most intriguing aspect of this study is the observation that all major changes in the qualitative pattern of protein synthesis take place between fertilization and the four- to eight-cell stage (day 3). From early day 3 onward, the qualitative pattern of protein synthesis remains essentially unchanged. Many of the major autoradiographic bands observed in mouse embryos from the four- to eight-cell stage and onward are also observed in protein patterns obtained from blastocyst-stage rabbit embryos. The changing patterns of protein synthesis revealed in this study occur before any gross differentiation of the embryos is evident (delineation of the inner cell mass and trophoblast) and before a marked increase in the relative rate of incorporation of L- [Y!i]methionine takes place. However, the qualitative changes in the pattern of protein synthesis do coincide with a period of extensive fine structural differentiation. INTRODUCTION
The preimplantation period of development of the mammalian embryo has been most extensively studied in the mouse. Studies describing changes in fine structure (Hillman and Tasca, 1969; McReynolds and Hadek, 1972), enzymatic activities (Biggers and Stern, 1973; Brinster, 1973; Graham, 1973a), metabolism and metabolic pathways (Biggers and Stern, 1973; Whitten, 1971), and macromolecular synthesis (Brinster, 1973; Graham, 1973a,b) have yielded a wealth of detailed information concerning early mouse embryogenesis. Results obtained from many of these studies are relatively unequivocal insofar as the reported observations are ‘Present address: B. F. Stolinsky Laboratory, Department of Pediatrics, University of Colorado Medical Center, Denver, CO 80220 and to whom reprint requests should be addressed. Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
148
generally in agreement. By contrast, results obtained by different investigators who have examined protein synthetic activities of preimplantation mouse embryos are in conflict. On the one hand, studies by Weitlauf and his collaborators (see Weitlauf (1974) for a summary) have been interpreted as indicating that no embryonic protein synthesis takes place in uiuo prior to blastocyst formation. On the other hand, experiments in which preimplantation mouse embryos have been exposed to radiolabeled amino acids in vitro suggest that protein synthesis occurs from the two-cell stage onward (Mintz, 1964; Brinster, 1973). Recently, Weitlauf (1974) attempted to resolve this discrepancy by suggesting that incorporation of labeled amino acids by cleaving embryos in vitro may primarily reflect “the turnover of existing protein and the synthesis of a small amount of a
VAN BLERKOM
AND BROCKWAY
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Synthesis
in Preimplantation
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specific protein involved in cleavage,” and microscopically to ensure that fertilization that the “increased incorporation shown had occurred and to determine the stage of after the morula and blastocyst stages in development and then placed into the vitro or at the blastocyst stage in uiuo” radioactive-labeling medium described bemay reflect the “beginning of a marked low. Eggs and follicle cells were obtained increase in the synthesis and accumulation from superovulated HS mice according to of new protein.” the procedure of Gates (1971). Follicle In the present communication, we pre- cells were removed from either eggs or sent results obtained from an analysis of early day 1 embryos by treatment with qualitative patterns of protein synthesis in hyaluronidase (100 international units follicle cells, ovulated, unfertilized eggs, (I.U.) per ml of culture medium, Worthingand cleavage- and blastocyst-stage em- ton Biochemical Corp.) for several minbryos. Because preimplantation mouse em- utes. Isotopic labeling of embryonic proteins. bryos do not incorporate labeled amino The culture medium used in these studies acids in uiuo until the blastocyst stage (Weitlauf, 1974), we have examined rates was that of Whitten (1971) with the follow(1) No protein suppleof incorporation and protein synthetic pat- ing modifications: terns of eggs and embryos labeled in vitro ment (such as bovine serum albumin) was with L- [YSlmethionine for short periods of present and (2) the organic buffer HEPES, time. Protein synthetic patterns were ob- N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Calbiochem) was added to tained by sodium dodecyl sulfate 20 mA4 at pH 7.4 (Van Blerkom and (SDS)-polyacrylamide-gel electrophoresis Manes, 1974). Radioactive culture medium in exponential gradient slab-gels followed was prepared by adding 0.475 ml of Whitby autoradiography (Van Blerkom and ten’s medium to 0.025 ml of L-[35S]methiManes, 1974). Our results indicate that not onine (New England Nuclear; specific aconly is the preimplantation period charactivity, 150 Ci/mmole) such that the final terized by a changing and complex pattern concentration of label in the medium was of protein synthesis but also that all major 200 pCi/ml. Radioactive methionine supqualitative changes in the pattern of proplied by the manufacturer is in water at 100 tein synthesis occur prior to approximately pCi/ml and contains 2-mercaptoethanol as the four- to eight-cell stage. a reducing agent. In a typical experiment. the 2-mercaptoethanol was removed, and MATERIALS AND METHODS the volume of the stock solution of labeled Embryo collection. Embryos were ob- methionine was reduced from 0.1 ml to 0.025 ml by lyophilization. tained from sexually mature, randomly Embryos were collected from the reprobred HS mice (Heterogeneous Stock deductive tract at three daily intervals, early, scribed by McClearn et al., 1970) following spontaneous ovulation. Females were mid, and late, during the entire preimplanplaced with males at 6 PM and checked for tation period. For eggs and cleaving embryos, culture in radioactive medium vaginal plugs a 8 AM the following morning. lasted 2 hr, according to the following Embryonic age is given as days following fertilization, with day 1 being the first day schedule: 8-10 AM (early), 2-4 PM (mid), and 9-11 PM (late). Blastocyst-stage emthe vaginal plug was detected. Animals were sacrificed by cervical dislocation, and bryos were exposed to label for 1 hr at the the oviducts and uteri were isolated and times indicated above. Between 25 and 40 embryos were labeled at one time for each either cut into small pieces in culture stage examined, and the labeling of each medium (oviducts) or flushed with culture embryonic stage was repeated at least six medium (uteri). Embryos were examined
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times. After exposure to label, all embryos appeared grossly normal. Ovulated, unfertilized eggs (with follicle cells removed) were labeled for 2 hr. Preparation of embryos for determinaand electrophoresis. tion of incorporation Following exposure to L- [35S]methionine, embryos were washed in several changes of warm, unlabeled medium (10 ml/wash). After the sequential washes, embryos or eggs were transferred in approximately 10 ~1 of medium to l-ml tubes containing 30 ~1 of lysis buffer (Van Blerkom and Manes, 1974). Both SDS and 2-mercaptoethanol are included in this buffer; to ensure complete lysis of the embryos, the samples were heated in boiling water for 1 min. The embryonic or egg samples were either used for electrophoresis or for determination of incorporation. Activities of samples (cpml ~1) were determined by precipitating the radiolabeled protein mixture (still in approximately 30 ~1 of lysis buffer) in 10% trichloroacetic acid (TCA), heating the mixture at 90°C for 10 min to hydrolyze any charged transfer ribonucleic acid, and then cooling the mixture at 5°C. The chilled samples were passed through Millipore filters with subsequent washes of cold 5% TCA and given a final rinse of cold 95% ethanol. The Millipore filters were dried, and retained radioactivity was measured under a toluene-PPO-POPOP scintillation mixture in a Beckman LS-250 counter. The relative rates of incorporation were determined from the activities of the samples, the number of embryos or eggs in each sample, and the duration of labeling. Follicle cells were labeled and prepared in the same manner as the embryos and eggs with the exception that the duration of labeling was 1 hr. Preparation and running of exponential acrylamide slab-gels. Exponential gradient gels containing 8-15% acrylamide were prepared as previously described Van Blerkom and Manes, 1974). Approximately 40,000 cpm was applied to each of the 15 separate wells on a slab. A typical exposure to X-ray
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film (Kodak No-Screen medical X-ray film, type NS2T) lasted 14 days. Molecular weights of embryonic proteins were estimated using both radioactive prereplicative bacteriophage T4 proteins and several other proteins such as the B, B’ subunits of E. coli RNA polymerase, RNase, lysozyme, myoglobin, bovine serum albumin, and chymotrypsinogen A (Van Blerkom and Manes, 1974). RESULTS
Quantitatiue
Aspects of Protein Synthesis
Incorporation of L- [YS]methionine into TCA-insoluble material by preimplantation mouse embryos measured at three daily intervals is shown in Fig. 1. The results are expressed as cpm per embryo per hr and represent values obtained from embryos exposed to label in vitro for a period between 1 and 2 hours (for each point). From the relative rates of incorporation, it is evident that, on a per embryo basis, no significant change occurs from early day 1 until some time between late day 3 and early day 4. In fact, there is a slight but reproducible decrease in incorporation between early day 1 and mid-to-late day 1. From late day 1 until approximately late day 3, mouse embryos incorporate about 500 cpmlhrlembryo when exposed to label of the same specific activity (150 Ci/mmole). An increase in incorporation is initially observed between late day 3 and early day 4 and continues to rise rapidly until implantation on mid day 5. The relative rate of incorporation on mid day 4 is approximately sixfold greater than previously and increases to 13-fold late on day 4. By mid day 5, embryos incorporate label at approximately 25 times the rate observed during cleavage. These values reflect only relative rates of incorporation, because measurements of uptake and endogenous pools of methionine were not undertaken. Qualitative Aspects of Protein Synthesis Qualitative changes in the pattern of embryonic protein synthesis throughout
VAN BLERKOM
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SO
Protein Synthesis in Preimplontation Mouse Embryos
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l \ *-.-o-o-.-.-.
ii 0
“““““““J El MI MI E2 Y2 M2 E3 M3 MS E4 M4 Y4 E5 &,LANTATION MI LI Y2 L2 MS L3 Y4 L4 M5 DAYS
FOLLOWING
FERflLlZATlON
1. The relative rates of incorporation of L- [%]methionine into TCA-insoluble material throughout the preimplantation period. Incorporation values were obtained at 3 daily intervals, early (E), mid (M), and late FIG.
ad. the entire preimplantation period are shown in Figs. 2 and 3. The electrophoretic separation and resolution of protein bands in this type of gel system (approximately 110 autoradiographic bands) is such that changes in the pattern of protein synthesis from one day to another are largely self-evident. As a consequence and in order not to increase the complexity of the autoradiograms, the actual identification of bands has been kept to a minimum. However, from a careful examination of each column, it is possible to obtain a general appreciation of the changing qualitative pattern of protein synthesis during early mouse embryogenesis. The qualitative pattern of protein synthesis in follicle cells, ovulated, unfertilized eggs, and early cleaving embryos is presented in Fig. 2. It is clear from this figure that follicle cells and unfertilized eggs are engaged in the synthesis of a complex pattern of proteins that range across the entire gel (and that have molecular weights ranging from approximately 8,000 at the bottom of the gel, to 250,000 at the top). It is also evident that proteins
synthesized by follicle cells are, for the most part, qualitatively different from proteins synthesized by unfertilized eggs. On the other hand, a significant degree of overlap exists between proteins synthesized by unfertilized eggs and day-l embryos. This overlap is especially evident in regions a-d in Fig. 2. It is also clear that day-l embryos synthesize proteins that are not being produced either by unfertilized eggs or follicle cells. The most outstanding examples of these proteins are located at approximately 35,000 daltons (arrows e) . What is most remarkable about these proteins is that not only do they represent a considerable portion of the total protein synthetic activity of day 1 embryos but also that the duration of their synthesis is less than 24 hr. Two characteristic features of the protein synthetic patterns of day-2 embryos are the appearance of a new set of autoradiographic bands and, of probably greater developmental significance, the disappearance of most bands observed in ovulated, unfertilized eggs and day-l embryos. Major new autoradiographic bands are evident in
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UNFERT;‘G;ED DAYS
El
LI
FOLLOWING
-
APPROXIMATE
I
E2
L2
FERTILIZATION l-2
NUMBER
2
2-4
OF CELLS
FPIG.2. The ! qualitative pattern (autoradiograph) of protein synthesis in follicle cells, ovulated, unfertiliz :ed eggs5 and early (El, E2) and late (Ll, L2) l- and e-day embryos. The approximate number of cells per embl :yo was determine sd after labeling. Approximate molecular weight values x lOA are given on the right.
E3L3
E4 M4
M4 L4
E5 M5
RABBIT BLASTOCYST
DAYS FOLLOWING FERTILIZATION 4-816
30
APPROXIMATE FIG. 3. The qualitative
60 NUMBER
100
-
OF CELLS
pattern (autoradiograph) of protein synthesis obtained from day-3, day-4 and day-5 embryos. No differences were observed when embryos labeled early (E), mid (M) and late (L) in each stage were compared. One qualitative difference in the pattern of protein synthesis in older embryos is evident on early-to-mid day 5; two bands located at approximately 12,000 daltons are not observed previously (arrow, E5-M5). The qualitative pattern of protein synthesis in 4.5-day-old rabbit embryo (blastocyst) is demonstrated in the column on the right, and bands common to mouse and rabbit embryos are indicated by arrows. The approximate number of cells per embryo was determined after labeling. Approximate molecular weight values x 10m3 are given at the right. 153
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regions f-j in Fig. 2. A comparison of early-to-mid day-2 patterns with late day-2 patterns reveals several qualitative differences. For example, several new proteins synthesized late on day 2 are observed in regions k-n. The final observed change in the qualitative pattern of protein synthesis during the preimplantation period occurs at some point between the four- and eight-cell stage (i.e., between early and mid day 3). Protein synthetic patterns obtained from day-3 and older mouse embryos are shown in Fig. 3. From approximately mid day 3 and for the remainder of the preimplantation period, the pattern of protein synthesis as revealed by this electrophoretic technique remains remarkably uniform and constant. Proteins synthesized by day-3 and older embryos are for the most part qualitatively different from proteins synthesized by earlier embryos. This qualitative difference is evident if Fig. 2 and 3 are closely compared. Of special interest in day-3 protein synthetic patterns are autoradiographic bands in the relatively low molecular weight region of the gel (S,OOO-15,000). Typically, these particular bands appear for the first time during the preimplantation period very early on day 3. The qualitative pattern of protein synthesis in a 4.5-day-old rabbit embryo is presented in the last column in Fig. 3. One feature that is quite clear from a comparison of mouse and rabbit protein synthetic patterns is that most of the major proteins produced by mouse embryos from day 3 onward are also synthesized by rabbit blastocysts. These particular bands are denoted by arrows in Fig. 3. Although the identity of the proteins composing these bands is unknown, preliminary studies indicate that in the mouse (as in rabbit blastocysts, Van Blerkom and Manes, 1974), actin is the major protein synthesized by embryos from day 3 onward (arrow a) and also by follicle cells. One additional protein, located between 50,000 and 55,000 daltons, has been tentatively identified as
VOLUME 44, 1975
tubulin (arrow t) (Van Blerkom, lished observations).
unpub-
DISCUSSION
Quantitative studies of the incorporation into TCA-insoluble of L- [35S]methionine material by preimplantation mouse embryos labeled in vitro indicate that no significant change occurs until some point between late day 3 and early day 4. From early day 4 until implantation on mid day 5, the rate of incorporation rises rather dramatically. The absolute rate of embryonic protein synthesis is a function of cell number, uptake, endogenous pool size, turnover rate, and the amino acid(s) used as a radioactive tracer (Epstein and Smith, 1973; Borland and Tasca, 1974). The relative rate of incorporation obtained in the present study should be interpreted with care as only incorporation into TCAinsoluble material was measured, and these values may not reflect the actual rate of embryonic protein synthesis. However, the rates of incorporation presented here are in general agreement with previous studies which indicated that no marked increase in incorporation occurs prior to approximately day 3 and that, after day 3, the rate of incorporation of radiolabeled amino acids into protein rises rapidly (Monesi and Salfi, 1967; Tasca and Hillman, 1970; Brinster, 1971, 1973; Epstein and Smith, 1973). Several cautions on the interpretation of autoradiographic patterns of embryonic protein synthesis were discussed in a previous paper (Van Blerkom and Manes, 1974). However, a few major points are worth restating here. With this type of gel system, approximately 110 autoradiographic bands can be resolved. For the most part, each band represents a heterogeneous collection of proteins that have the same approximate molecular weight. Thus, proteins separated by electrophoresis and visualized by autoradiography are those that would be either individually or collectively present in relatively high concentrations.
VAN BLERKOM
AND BROCKWAY
Protein Synthesis in Preimplantation
With these cautions on interpretation in mind, several qualitative aspects of embryonic protein synthesis are evident from an analysis of the autoradiographs. Most proteins synthesized by day-l embryos (and resolved in this gel system) also appear to be synthesized by ovulated, unfertilized eggs but not by follicle cells. By day 2, most of these “early” proteins are no longer being produced or are synthesized at a greatly reduced level (i.e., by the two-cell stage). If proteins synthesized by ovulated eggs are considered to be “maternal” proteins, then it is quite evident that the newly fertilized egg continues to produce primarily maternal proteins. That newly fertilized eggs are not synthesizing exclusively maternal proteins is clear from an examination of day-l protein synthetic patterns. Several proteins synthesized by day-l embryos are not observed in patterns obtained from unfertilized eggs. The major examples of these proteins are located at approximately 35,000 daltons and represent a significant percent of the total protein synthetic activity of the early embryo. Protein synthetic patterns of day-2 embryos reveal several interesting features of this stage of development. First, most proteins synthesized on day 1 (and by eggs) are no longer being produced on day 2. Most of the day-2 proteins resolved in this system are synthesized for approximately 24 hr or less. Secondly, qualitative differences are evident when early and late day-2 protein synthetic patterns are compared. Although these differences are reproducible, their developmental significance is unknown. The qualitative pattern of protein synthesis observed in embryos from early to mid day 3 (four- to eight-cell stage) and from that point onward is remarkable for the following reasons: (1) The qualitative patterns are significantly different from what was observed previously, and (2) no further qualitative changes in the pattern of protein synthesis take place for the
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remainder of the preimplantation period. Many “new” proteins are synthesized by day-3 embryos, and, of these, several are of particular interest because they represent the major species of proteins synthesized by embryos from about the four-cell stage onward. A comparison of mouse protein patterns in day-3 and older embryos with 4.5-day-old rabbit embryos indicates a striking degree of similarity. The bands that contain the major species of proteins in day-3 and older mouse embryos are also the major bands of proteins in rabbit blastocysts. The major protein synthesized by mouse and rabbit embryos (and follicle cells) appears to be actin (Van Blerkom and Manes, 1974). Although the identity of the other major proteins common to mouse and rabbit embryos is unknown, one possibility is that they represent proteins involved in the maintenance of the structural integrity of the embryo and are required for the formation of the blastocyst (Van Blerkom and Manes, 1974). At present, it is quite difficult to interpret the developmental significance of the qualitative changes in the pattern of protein synthesis. This is true not only because the proteins that compose the autoradiographic bands are unidentified but also because little is known relevant to their origin (maternal vs embryonic mRNA). There are, however, several possibilities that may be helpful in attempting to understand what these patterns may mean in the course of early mouse embryogenesis. Numerous changes in enzyme constitution and activity of preimplantation mouse embryos have been studied in detail (Biggers and Stern, 1973; Brinster, 1973). Although it is doubtful whether most enzymes would be present in the embryo in amounts sufficient to be resolved individually in a study of whole embryos, enzymatic changes cannot be excluded from consideration. Alternately, the synthesis of many proteins may depend upon the participation of the paternal genome. However, studies with partheno-
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genie cleavage and blastocyst-stage mouse embryos demonstrate that the qualitative pattern of protein synthesis in these embryos is essentially identical to patterns obtained from normally fertilized eggs at the same stage of development (Van Blerkom and Runner, unpublished observations) . Although the qualitative pattern of protein synthesis remains invariant during the period of gross morphologic differentiation (i.e., blastocyst formation and the elaboration of the inner cell mass and trophoblast), many of the observed changes occur during a period of rapid fine structural differentiation. Comprehensive studies of the fine structural differentiation of the preimplantation mouse embryo (Calarco and Brown, 1969; Hillman and Tasca, 1969) have demonstrated that the development and differentiation of major organelle systems are initiated at fertilization and are essentially complete by the eight- to twelve-cell stage. Major fine structural changes that occur during this period include the following: (1) An increase in the number of cytoplasmic vesicles, crystalloid bodies and ribosomes, (2) differentiation and activation of mitochondria and nucleoli, and (3) elaboration of the rough-surfaced endoplasmic reticulum and formation of junctional complexes. Possibly, the changing patterns of protein synthesis observed during early mouse development are correlated with fine structural differentiation known to occur during this period. A similar correlation between fine structural differentiation and changing qualitative patterns of protein synthesis has been demonstrated during preimplantation development in the rabbit (Van Blerkom et al., 1973; Van Blerkom and Manes, 1974). In the rabbit, as in the mouse, the major qualitative changes in the pattern of protein synthesis occur during cleavage when most of the major fine structural alterations are initiated and, in most cases, completed. This observation is rather surprising in light of the incorporation results
VOLUME 44, 1975
presented earlier and indicates that the increase in the relative rate of incorporation of radiolabeled amino acid(s) occurs both after the fine structural differentiation of the embryo is largely completed and after most changes in the qualitative pattern of protein synthesis have occurred. An additional point to be considered concerns the nature of differential gene expression in preimplantation development. As is evident from a detailed examination of both mouse and rabbit protein synthetic patterns (Van Blerkom and Manes, 1974), many proteins are synthesized for only a relatively short period of time, and, of these, some are produced in significant amounts. It has become increasingly apparent from these studies that early mammalian development includes not only the progressive “turning on” of genetic information, but also, and perhaps of equal importance, a relatively rapid “turning off” of this information and halt in the synthesis of specific proteins. Perhaps both the turning on of genes and the scheduled shut-off of their expression represent functions that are an integral part of an overall autonomous developmental program contained within the genome. Finally, as mentioned earlier, results obtained from quantitative studies of early embryonic protein synthesis in vivo and in vitro are in conflict (Weitlauf, 1974). Although our results do not clarify this discrepancy, they do demonstrate that, in a qualitative sense, the entire preimplantation period is characterized by a complex and changing (at least in the early phases) pattern of protein synthesis. Experiments currently in progress should establish whether these autoradiographic patterns, obtained from embryos labeled in vitro for relatively brief periods of time, reflect actual in vivo protein synthetic activity. Addendum. After this paper was submitted, a study by C. J. Epstein and S. A. Smith of qualitative patterns of protein synthesis by preimplantation mouse embryos appeared in Developmental Biology
VAN BLERKOM AND BROCKWAY
Protein
Sj nthesis in Preimplantation
(Develop. Biol. 40, 233-244 (1974)). Results obtained in that study are in general agreement with the results presented in this communication. We express our appreciation to Dr. Cole Manes for his continued support and helpful criticisms. Funds for this work were made available from a grant to Dr. Cole Manes (Grant No. HD-04274 from the National Institutes of Health, United States Public Health Service). REFERENCES BIGGERS, J. D., and STERN, S. (1973). Metabolism of the preimplantation mammalian embryo. In “Advances in Reproductive Physiology” (M. W. H. Bishop, ed.) Vol. 6, pp. 1-59. Paul Elek Scientific Books, London. BORLAND, R. M., and TASCA, R. J. (1974). Activation of a Na+-dependent amino acid transport system in preimplantation mouse embryos. Deuelop. Biol. 36, 1699182. BRINSTER, R. L. (1971). Uptake and incorporation of amino acids by the preimplantation mouse embryo. J. Reprod. Fert. 27, 329-338. BRINSTER, R. L. (1973). Protein synthesis and enzyme constitution of the preimplantation mammalian embryo. In “The Regulation of Mammalian Reproduction” (S. Segal, R. Crozier, P. A. Corfman, and P. G. Condliffe, eds.). pp. 3022334. C. C Thomas, Springfield, IL. CALARCO, P. G., and BROWN, E. H. (1969). An ultrastructural and cytological study of preimplantation development of the mouse. J. Exp. Zool. 171, 253-283. EPSTEIN, C. J., and SMITH, S. (1973). Amino acid uptake and protein synthesis in preimplantation mouse embryos. Deuelop. Biol. 33, 171-184. GATES, A. H. (1971). Maximizing yield and developmental uniformity of eggs. In “Methods in Mammalian Embryology” (J. C. Daniel, ed.), pp. 64-75. W. H. Freeman, San Francisco. GRAHAM, C. F. (1973a). The necessary conditions for gene expression during early mammalian development. In “Genetic Mechanisms of Development” (31st Symppsium, The Society for Developmental
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Biology) (F. H. Ruddle, ed.), pp. 202-224. Academic Press, New York. GRAHAM, C. F. (1973b). Nucleic acid metabolism during early mammalian development. In “Regulation of Mammalian Reproduction” (S. Sepal, R. Crozier, P. A. Corfman, and P. G. Condliffe, eds.), pp. 286-301. C. C Thomas, Springfield, IL. HILLMAN, N., and TASCA, R. J. (1969). Ultrastructural and autoradiographic studies of mouse cleavage stages. Amer. J. Anat. 126, 151-174. MCCLEARN, G. E., WILSON, J. R., and MEREDITH, W. (1970). The use of isogenic and heterogenic mouse stocks in behavioral research. In “Contributions to Behavior-Genetic Analysis-The Mouse as a Prototype” (G. Lindzey and D. D. Thiessen, eds.), pp. 3-22. Appleton-Century-Crofts. MCREYNOLDS, H. D., and HADEK, R. (1972). A comparison of the fine structure of late mouse blastocysts developed in uiuo and in uitro. J. Exp. Zool. 182, 95-118. MINTZ, B. (1964). Synthetic processes and early development in the mammalian egg. J. Exp. Zool. 157, 85-100. MONESI, V., and SALFI, V. (1967). Macromolecular synthesis during early development in the mouse embryo. Exp. Cell Res. 46, 632-635. TASCA, R. J., and HILLMAN, N. (1970). Effects of actinomycin D and cycloheximide on RNA and protein synthesis in cleavage stage mouse embryos. Nature (London) 225, 1022-1025. VAN BLERKOM, J., ~~~MANEs, C. (1974). Development of preimplantation rabbit embryos in uiuo and in uitro. II. A comparison of qualitative aspects of protein synthesis. Develop. Biol. 40, 40-51. VAN BLERKOM, J., MANES, C., and DANIEL, J. C., JR. (1973). Development of preimplantation rabbit embryos in uiuo and in uitro. I. An ultrastructural comparison. Deuelop. Biol. 35, 262-282. WEITLAUF, H. M. (1974). Metabolic changes in the blastocysts of mice and rats during delayed implantation. J. Reprod. Fert. 39, 213-224. WHITTEN, W. K. (1971). Nutrient requirements of the culture of preimplantation embryos in uitro. In “Advances in the Biosciences” (G. Rasp& ed.), Vol. 6, pp. 129-141. Pergamon Press, London.
