Effect of metabolic inhibitors on macromolecular synthesis and early development in the mouse embryo

Experimental

EFFECT SYNTHESIS

OF METABOLIC AND

EARLY

V. MONESI, CNEN, Laboratorio

Cell Research 59 (1970) 197-206

INHIBITORS DEVELOPMENT

M. MOLINARO,

ON MACROMOLECULAR IN THE

E. SPALLETTA

MOUSE

EMBRYO

and C. DAVOLI

di Radiobiologia animale CSN Casaccia, Rome, and Institute of Histology and General Embryology of the University of Rome, Italy

SUMMARY Mouse embryos were cultured in vitro for various periods of time, during the interval from the 2-cell stage to the late blastocyst stage, in the continuous presence of actinomycin D or puromycin and labeled precursors. At various time intervals, the incorporation of *H-uridine into total RNA and of 3H-leucine into protein, and the stage of development of the embryos were recorded. Actinomycin D at the concentration of 0.1 pg/ml caused a rapid and almost complete inhibition of the incorporation of 8H-uridine at all stages of development, and a rapid depression of the incorporation of SH-leucine into protein until a level of about 50 % of the control incorporation which was attained after 12-16 h of incubation. Longer incubation with the antibiotic did not further depress the relative incorporation of “H-leucine with respect to the control. The development of the embryos in culture was markedly depressed after continuous incubation with 0.01 and 0.1 ,ug/ml of actinomycin D, but was not completely arrested. Puromycin at the concentration of 50 pg/ml caused an immediate and complete inhibition of SH-leucine incorporation and of development in culture. These results were interpreted to indicate that in the mouse embryo protein synthesis and normal development in culture from the 2-cell stage to the blastocyst stage are regulated by a continuous synthesis of RNA. These results correlate with previous biochemical evidence that in the mouse embryo, genes are transcribed very early during development. There is, however, a large fraction of protein synthesis which seems to be dependent on RNA molecules with very long half-life; this fraction of protein synthesis accounts probably for the partial development occurring after actinomycin treatment. The culture methods available at present do not allow to establish whether these stable messengers are synthesized during oogenesis or after fertilization. The early dependence of embryonic development on gene activity in the mouse contrasts then markedly with the situation observed in sea urchin and amphibians, where embryonic development and protein synthesis until gastrulation are completely independent of simultaneous gene activity, but are probably fully regulated by ribosomes and stable RNA messengers synthesized during oogenesis and stored in the egg cytoplasm to be utilized after fertilization.

The study of nucleic acid and protein synthesis and the analysis of the effect of metabolic inhibitors during early development may provide information on the control mechanisms that operate during embryogenesis. Recently, there has been a growing accumulation of information concerning the biochemistry of early development in echinoderms and amphibia. By contrast, the present knowledge on the regulation of macromolecular synthesis in mammalian embryos is still scarce.

The study of macromolecular synthesis in mammalian embryos has recently been facilitated by the availability of media that permit development in vitro of mouse eggs until the blastocyst stage and by the use of hormonally induced superovulation in the mouse. The available information indicates that in mammalian embryos the synthesis of RNA is activated very early after fertilization, as compared to non-mammalian embryos. Previous autoradiographic studies have shown Exptl Cell Res 59

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that in the mouse the incorporation of labeled precursors into extranucleolar and nucleolar RNA occurs very soon after fertilization, at the 2- and 4-cell stage respectively, and increases steadily afterwards with a pronounced acceleration from the morula to the blastocyst stage [15, 18, 191. 3H-Leucineincorporation occurs continuously throughout cleavage and blastulation but shows a pronounced acceleration from the 8-cell stage until the blastocyst stage [19]. The biochemical analysis has given a better quantitative description of the pattern of macromolecular synthesis during early development in the mouse. Monesi & Salfi [21] have shown that incorporation of 3H-uridine into total RNA is absent in the unfertilized tubal egg and remains very low until the 12-16-cell stage. After the 12-16-cell stage until the morula and blastocyst stage the rate of incorporation increases sharply up to about 90 times the initial value. Unlike RNA synthesis, some incorporation of 3H-leucine and 3H-lysine into protein occurs also before fertilization. After fertilization, the rate of protein synthesis remains rather low until the 8-cell stage and increases very rapidly afterward, a little in advance of the time of the rise of RNA synthesis [21]. Recently, Woodland & Graham [30], by sucrose gradient sedimentation analysis, have demonstrated that in the mouse the synthesis of 28 S and 18 S ribosomal RNA and of 4 S RNA occurs very early after fertilization, that is during the late 4-cell stage. Prior to the 4-cell stage, small amounts of high molecular weight and low molecular weight RNA are synthesized by the embryos. Ellem & Gwatkin [l I], using MAK chromatography, have detected synthesis of ribosomal RNA, of low molecular weight RNA and DNA-like RNA at the 8-cell stage in the mouse embryo and a rapidly increasing rate of synthesis until morula and blastocyst stage. Results similar to Exptl Cell Res 59

those published by Woodland & Graham have been obtained by us (fig. 2). The biochemical evidence of an early activation of ribosomal RNA synthesis during embryogenesis in the mouse is further supported by the electron microscopic observations on the evolution of the nucleolus and ribosomes. These studies have shown the scarcity of ribosomes and a prevalently fibrillar organization of the nucleolus until the 4-8cell stage [15, 171. After this stage, the nucleolus begins to exhibit the 150 A granular component [15, 171, which is interpreted as expression of accumulation of ribosomal precursors, and the ribosomes begin to accumulate in the cytoplasm [17]. The effect of actinomycin on development and nucleic acid and protein synthesis in mammalian embryos has been scarcely studied quantitatively. However, a few observations indicate that the mouse embryo is very sensitive to actinomycin D: at low concentrations the antibiotic blocks the development of the embryo in culture at early stages [19, 26, 281. Using autoradiography, Mintz [19] has reported that in the mouse the nucleolar RNA labeling is completely inhibited by low doses of actinomycin (1O-7 M) which cause a rapid arrest of cleavage in culture, whereas protein synthesis and also extranucleolar RNA synthesis persist to a large extent even at much higher concentrations of the antibiotic. Ellem & Gwatkin [l I] have also found that 1 h treatment of mouse blastocysts in culture with 1O-7 M actinomycin D causes a gross inhibition of rRNA and sRNA synthesis but has only a slight effect on the synthesis of DNA-like RNA. MATERIAL

AND METHODS

The embryos were obtained from 8- to 12-week-old random-bred Swiss mice after spontaneous ovulation. At 5 p.m. the females were placed with males and checked for the presence of the vaginal plug the following morning at 9 a.m. At fixed times after detection

Effect of metabolic inhibitors in mouse embryo of the copulation plug the animals were killed, the Fallopian tubes were isolated and their content was flushed out with saline solution. The embryos were microscopically checked to determine the stage of development and incubated in approx. 0.1 ml of culture medium at 37°C with 5 % CO* in air under liquid paraffin. The distinction between early (about 32 cells), middle and late (32 to 80 cells) blastocyst was not based on cell counts but on the relative size of the inner-cell mass and the segmentation cavity.

Culture methods The embryos obtained from several females were pooled together and then distributed in groups, 30 to 50 embryos per group. Each sample of 30 to 50 embryos-was-incubated in approx. 0.1 ml of culture medium containing the radioactive precursor at 37°C with 5 % CO, in air under 2 ml of liquid paraffin, for different periods of time as indicated in the figures, and then counted for the incorporated radioactivity. The rate of development of the embryos in culture (figs 6,7,9) was determined on a single sample of 50 to 100 embryos by estimating microscopically at various intervals of incubation the developmental stage of each embryo and plotting the average number of blastomeres per embryo. The culture medium used in these experiments was a modification by Mulnard [23] of Brinster’s medium [l-3], with the addition of antibiotics and the substitution for human albumin of an equal quantity of bovine albumin. The composition of this medium was as follows: 7.100 g/l NaCl; 0.350 g/l KCI; 0.184 g/l CaCI,; 0.130 g/l NaHaPO1*HaO; 0.090 g/l MgS04; 1.OOOg/l NaHCO,; 1.326 g/l sodium lactate; 0.013 g/l sodium pyruvate; 5.000 g/l bovine serum albumin (Sigma); 0.020 g/l phenol red; 100 U/ml penicillin; 50 fig/ml streptomycin. The rate of development of the control embryos in culture is presented in figs 6 and 9.

Radioactive precursors and metabolic inhibitors The concentrations in the culture medium of the radioactive precursors were: 40 ,&i/ml *H-5-uridine (24 C/mM, Radiochemical Centre, Amersham); 10 ,&i/ml aH-4,5-L-leucine (5 Ci/mM, New England Nuclear Corp.). The metabolic inhibitors were actinomycin D used at the concentrations of 1O-8, 1O-7 or 1O-B M and puromycin at the concentration of 50 ,ug/ml in culture medium. The radioactive compounds and the inhibitors were present in the culture medium throughout the period of incubation. At the end of the incubation period, the embryos were thoroughly washed in three changes of nonradioactive medium containing the unlabeled precursor 1000 times more concentrated than the radioactive compound in the culture medium. Afterwards, the embryos were microscopically checked to determine the stage of development, counted and squashed on a disk of Whatman no. 3MM chromatography paper 1.5 cm in diameter. After air drying, the disks were accumulated in 99°C ethylalcohol at 0°C

199

until the time of counting. The precipitation-extraction procedure was carried out as follows. The disks were successively washed in two changes of 10% trichloracetic acid (TCA) for 30 min ai WC, three raoid changes of cold TCA and 3 % cold oerchloric acid (PCA), cold ethanol for 10 min, e&y1 etherethanol mixture 1: 1 at 37°C for 30 min, cold ethyl ether for 5 min, ethyl-sther at room temperature for 5 min. and then air dried. Air dried disks were then placed in counting vials containing 10 ml of scintillation liquid (PPO 4 g and POPOP 40 mg in 1 1 toluene). The radioactivity was determined in a model 3000 Tri-Carb Packard liauid scintillation soectrometer. The background was hetermined in blanks disks prepared by pipetting on filter-paper disks an aliquot of the cold medium used for washing the embryos, and carried through the precipitation-extraction-washing procedure with the sample disks. The background was 10-20 cpm per disk and was subtracted from the counts.

RESULTS RNA synthesis

Fig. 1 shows the rate of incorporation of 3Huridine by embryos explanted at different stages of development and incubated in culture with various concentrations of actinomycin D for the periods of time indicated in the figure. The figure indicates that very little 3Huridine incorporation into RNA occurs after treatment with 0.1 pug/ml of actinomycin, whereas the control embryos show a continuous incorporation of radioactivity. The residual incorporation after actinomycin treatment probably does not reflect net synthesis of RNA but might be due to the turnover of the terminal CCA sequence of tRNA. Since these experiments were carried out in the continuous presence of the antibiotic and the radioactive precursor, the specific activity of the pool presumably remains constant throughout the time of incubation, after the first equilibration period. This would explain why the total radioactivity due to end terminal addition to tRNA does not decline during the period of incubation. It cannot, furthermore, be excluded that some of the incorporation occurs into DNA through conversation of uridine into deoxycytidine. Exptl

Cell Res 59

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V. Monesi et al.

.

jl-cellsiq

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6

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0 )

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1

2

3

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5

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Abscissa: incubation period (hours); ordinate: SH-uridine cpm/embryo. Fig. I. SH-Uridine incorporation into total RNA by mouse embryos explanted at 2-cell, 4-cell, g-cell, or morula and blastocyst stages, and cultured in vitro for various intervals of time in the continuous presence of the radioactive precursor and of actinomycin D.

The lack of effect of actinomycin D on extranucleolar labeling shown previously by Mintz [19] and on the synthesis of DNA-like RNA observed by Ellem & Gwatkin [I l] might be due to the very short duration (30 min to 1 h) of the treatment with the antibiotic in these authors’ experiments. It is in fact known that the synthesis of ribosomal RNA is affected to a much larger extent by low concentrations of actinomycin D than the transcription of other RNA classes [24, 251.

Fig. 1 shows that the rate of incorporation of 3H-uridine into total RNA in the control embryos increases markedly during early deExptl Cell Res 59

velopment with a dramatic acceleration (of about 50-fold) from the &cell stage to the morula and blastocyst stage. The precursor incorporation into RNA increases during cleavage at a much higher rate than the number of blastomeres per embryo. In principle, the observed change in the rate of incorporation of the precursor during early development cannot be equated with a change in rate of net RNA synthesis unless it is demonstrated that the specific activity of the precursor pool does not vary during development. No attempt was made in these experiments to measure the specific activity of the pool. Woodland & Graham [30] have seen

Effect of metabolic inhibitors in mouse embryo

that the total acid soluble radioactivity does not vary significantly between the 2- and the g-cell stage embryo, but have also failed to measure the specific activity of the pool, owing to obvious technical difficulties with this material. The sedimentation analysis by sucrosedensity-gradient centrifugation has shown a significant incorporation of 3H-uridine in the 18 S and 28 S regions of the gradient at as early as the 4-cell stage embryo (fig. 2). This finding is similar to the results reported by Woodland & Graham [30] showing that the synthesis of ribosomal RNA begins at the 4cell stage in the mouse embryo. Protein synthesis

Figs 3 and 4 show the effect of actinomycin on 3H-leucine incorporation in experiments of continuous incubation with the antibiotic and the radioactive precursor. Fig. 3 reports the rate of incoporation, expressed as total counts per embryo, as a function of time of incubation. This figure shows that, following actinomycin treatment, the rate of incorporation is greatly depressed, at all stages of development. However, a significant incorporation is still detectable at a dose of the antibiotic (0.1 lug/ml) which completely inhibits the synthesis of RNA. At a lower concentration of actinomycin (0.01 pg/ml), the inhibition is less pronounced which is accounted for by the residual synthesis of RNA detected at this dose. In order to obtain a more clear picture of the rate of depression of the incorporation, the results from several experiments were pooled together and expressed as a percentage of the incorporation of the control at each interval of incubation (fig. 4). Fig. 4 shows that the inhibition of protein synthesis by 0.1 pg/ml of actinomycin occurs very early after treatment. During the first three cleavages the percentage incorporation with re-

201

tube no., the top of the gradient on the left; cpm ( l ) and A,,, x lOa (0). Sucrose-density-gradient centrifugation of RNA extracted from 4-cell stage embryos labeled with JH-5-uridine (100 &i/ml) for 6 h in culture. The OD trace represents added carrier RNA from Chinese hamster cells. The cpm trace refers to acid-precinitated RNA on Millinore filter. The figure indicates that a significant incorporation of radioactivity in the 18s and 28s regions of the gradient occurs in the 4-cell stage embryo. Abscissa: ordinate: Fig. 2.

spect to the control drops rapidly up to a minimum of about 50% which is attained after 12-16 h incubation with the antibiotic, and remains approximately constant thereafter. This plateau of relative inhibition was not detected at the morula and blastocyst stages since these experiments were interrupted after 8 h of incubation, because of the technical difficulties of culturing these late stages for longer periods of time. The dose-effect curve for actinomycin is represented in fig. 5. This figure shows that in the 8-cell stage embryo the 3H-uridine incorporation decreases to about 20 % of the control rate after 18 h incubation with a dose of 0.01 ,ug/ml of actinomycin and drops to about 2 % with 0.1 ,ug/ml. The 3H-leucine curve shows a modest inhibition at the lower dose of actinomycin and a further depression of the relative incorporation with increasing doses up to about 40% of the control. The residual incorporation of 3H-leucine at the concentration of 0.01 mg/ml is accounted for Exptl

Cell Res 59

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Morulo ond BLostMyst 3504

lW-

CONTROL

600500. ACT.O~O.ly/mll

0

5

10

15

20

25

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Abscissa: incubation period (hours); ordinate: 8H-leucine cpm/embryo. Fig. 3. SH-Leucine incorporation into protein by mouse embryos explanted at 2-, 4- &cell stage or morula and blastocyst stage and incubated in vitro for various periods of time in the continuous presence of the radioactive precursor and of actinomycin D.

by the residual synthesis of RNA. The 40 % protein synthesis remaining after the almost complete inhibition of RNA synthesis caused by higher doses is probably dependent on long-lived messengers (see below). As expected, the 3H-leucine incorporation is immediately and completely inhibited by incubation with 50 ,ug/ml of puromycin (fig. 8). Development

The pattern of development in culture in the presence of actinomycin is consistent with the severe reduction of protein synthesis. ExptI Cell Res 59

Fig. 6 shows that the continuous incubation with actinomycin D causes a marked retardation of development in culture. During a period of 24 h the 2-cell stage embryos undergo one cleavage, as an average, in the control, whereas only a fraction of the treated embryos undergo one division; the 4- and &cell stage embryos undergo two cleavages in the control but only one after actinomycin treatment. The inhibitory effect of the drug is even more severe at the 16-cell, morula and blastocyst stages (fig. 7). Figs 8 and 9 show that leucine incorporation and development in culture are immedia-

Effect of metabolic inhibitors in mouse embryo

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Abscissa: incubation period (hours); ordinate: SH-leucine incorporation (cpm) percent of the control. Fig. 4. 3H-leucine incorporation expressed as percent of the control at each interval of time of culture, after incubation for various periods with actinomycin D. tely and completely inhibited after incubation with 50 ,ug/ml of puromycin. These results are interpreted to indicate that the partial development observed after actinomycin treatment does not depend on a storage of proteins related to cell division synthesized prior to the beginning of the treatment.

DISCUSSION Previous biochemical observations have demonstrated that in the mouse embryo genes are transcribed very early during development [l 1, 21, 301. The best evidence of an early activation of genes in the mouse embryo conExptl Cell Res 59

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Abscissa: concentration of actinomycin D; ordinate: percent of the control incorporation. Fig. 5. Inhibition of 8H-leucine and 8H-uridine incorporation in 8-cell stage embryos incubated for 18 h with various concentration of actinomycin D. The incorporation is expressed as percent of the cpm/ embryo with respect to the control.

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terns the transcription of the ribosomal cistrons which occurs in the late 4-cell stage embryo ([30] and the present communication). The present findings on the effect of actinomycin D show that the ribonucleic acids synthesized during cleavage are immediately and continuously required to sustain protein synthesis and development from the 2-cell stage to the late blastocyst stage. This early dependence of embryonic development on gene activity in the mouse contrasts then markedly with the situation observed in sea urchin and amphibia where embryonic development until gastrulation is completely independent of gene activity but is probably fully supported by stable messengers and ribosomes synthesized during oogenesis and stored in the egg cytoplasm to be utilised after fertilization [5, 12-14, 22, 291. However, since in the mouse, following actinomycin treatment for as long as 24 h, some 50 % of protein synthesis and a partial Exptl Cell Res 59

4

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Abscissa: incubation period (hours); ordinate: cell stage. Fig. 6. Development in culture of 2-, 4- and 8-cell stage mouse embryos in the absence (control) or in the continuous presence of actinomycin D. The rate of development was determined by estimating microscopically at each interval of incubation the stage of development of each embryo and plotting the average number of blastomeres per embryo. O-O, control; U---U, actinomycin D (0.01 pg/ml); O-O, actinomycin D (0.1 ,ug/ml).

development still occur, it seems reasonable to suggest that in the mouse early embryogenesis is also partially regulated by stable messengers. Since in most experiments the

Effect of metabolic inhibitors in mouse embryo

period of incubation has been limited to a maximum of 24 h (because of the difficulties of obtaining a good development in culture with longer incubation times), it cannot be established as to whether these stable messengers are synthesized during oogenesis or after fertilization. This problem could be solved if experimental conditions were available to sustain embryonic development in culture from fertilization to blastulation. The culturing methods available at present give unfortunately a rather poor yield of development after long periods of incubation which is unsatisfactory for biochemical studies, and the methods of fertilization of mouse eggs in vitro need further improvement to be amenable for biochemical work. An important problem in the study of regulation of embryonic development in the mouse is that of the relative role of the different classes of RNA synthesized during cleavage. In fact, since in the late 4-cell stage embryo the ribosomal cistrons are abruptly activated, the effect of actinomycin on protein synthesis and development in the 4- and 8-cell stage embryos might be partially ascribed to the inhibition of transcription of ribosomal genes. An effective method of approaching this problem experimentally might be provided if an anucleolate mutant similar

205

Abscissa: incubation period (hours); ordinate: percentage. Fig. 7. Histogram of development in culture of mouse embryos, explanted at 16-cell, 32-cell, or early blastocyst stage (0 time) and incubated for 12 or 8 h in the absence (C) or in the presence of 0.1 pg/ml of actinomycin D (0.1). EE, early blastocyst; M, middle blastocyst; LB, late blastocyst. The distinction between early (about 32 cells) middle and late (32 to 80 cells) blastocysts was not based on cell counts but on the relative size of the inner-cell mass and the

segmentation cavity.

to that found in Xenopus laevis [4-81, existed also in the mouse. Many authors have described in the mouse a recessive mutation at the complex T locus known as T12 [9, lo], which in the homozygous condition is characterized by abnormal morphology of nucleoli [19, 20, 271, lower concentration of

a

incubation period (hours); ordinate: *H-leucine cpm/embryo. Incorporation of BH-leucine by embryos explanted at (a) 2- and (b) 8-0~11 stage and incubated in culture in the absence or in the continuous presence of 50 pg/ml of puromycin. The radioactive precursor was continuously present in the culture medium. O-O, control; O-O, puromycin (50 pg/ml). Abscissa: Fig. 8.

Exptl

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Abscissa: incubation period (hours); ordinate: cell stage. Fig. 9. Development in culture of (a) 2- and (b) 4-cell stage embryos in the absence or in the continuous presence of 50 pg/ml of puromycin. See fig. 6 for the description of the method of determination of the rate of development. O-O, control; O-O, puromycin (50 pg/ml).

cytoplasmic and nucleolar RNA [19-20, 271, diminished incorporation of 3H-uridine [20], and arrest of development at the morula stage [19,20,26,27]. If it can be demonstrated that this mutant has a deletion of ribosomal genes, as suggested byhybridization experiments on adult heterozygotes [16], then this system could represent a very useful tool to study the relative role of the different classes of RNA in the regulationof cleavage. This research work has been supported in part by PHS grant no. ROl-EC00086 from the Division of Radiological Health, USA, and grant no. 69.02.193 from the Consiglio Nazionale delle Ricerche.

REFERENCES 1. Brinster, R L, Exptl cell res 32 (1963) 205. 2. -J reprod fertil 10 (1965) 227. 3. Brinster, R L & Thomson, J L, Exptl cell res 42 (1966) 308. 4. Brown, D D, J exptl zoo1 157 (1964) 101. 5. - Current tooics in develonmental bioloav (ed A A Moscona & A Monroy) vol. 2. A&de&c Press, New York (1967). 6. Brown, D D & Gurdon, J B, Proc natl acad sci IJS 51 (1964) 139. 7. - J mol biol 19 (1966) 399. 8. Brown, D D & Littna, E, J mol biol 8 (1964) 669.

Exptl Cell Res 59

9. Dunn, L C, Cold Spring Harbor symp quant biol 21 (1956) 187. 10. Dunn, L C & Gluecksohn-Waelsch, S, Genetics 38 (1953) 261. 11. Ellem, K A 0 & Gwatkin, R B L, Develop bioll8 (1968) 311. 12. Gross, P R, Current topics in developmental biology (ed A A Moscona & A Monroy) vol. 2. Academic Press New York (1967). 13. Gross, P R & Cousineau, G H, Exptl cell res 33 (1964) 368. 14. Gross, P R, Malkin, L I & Moyer, W A, Proc natl acad sci US 51 (1964) 407. 15. Hillman. N W & Tasca, R J. Proc XII international congress genetics, Tokyo, Abst 7.5.1. (1968) 16. Klein, J & Raska, K, Proc XII International genetics, Tokyo, Abst 7.5.3. (1968). 17 congress Maraldi, N & Monesi, V, Z Zellforsch. Submitted ’ publication (1970). 18 for Mintz, B, Am zoo1 2 (1962) 432. 19: - J exptl zoo1 157 (1964) 85. 20. - Ibid 157 (1964) 273. 21. Monesi, V & Salfi, V, Exptl cell res 46 (1967) 632. 22. Monroy, A, Arch biol 76 (1965) 511. 23. Mulnard, J G, Arch biol 78 (1967) 107. 24. Perrv, R P. Proc natl acad sci US 48 (1962) 2179. 25. - Exptl cell res 29 (1963) 400. 26. Silagi, S, Exptl cell res 32 (1963) 149. 27. Smith, L J, J exptl zoo1 132 (1956) 51. 28. Thomson. J L & Biazers. J D. Exotl cell res 41 (1966) 41i. -’ ’ 29. Tyler, A, Develop biol. Suppl 1 (1967) 170. 30. Woodland, H R & Graham, C F, Nature 221 (1969) 327. Received June 25, 1969 Revised version received October 6, 1969