Uridine and Guanosine Incorporation by the Mouse One-Cell Embryo

/. Embryol. exp. Morph. Vol. 44, pp. .133-148, 1978 \ 33 Printed in Great Britain © Company of Biologists Limited 1978

Uridine and guanosine incorporation by the mouse one-cell embryo

By R. J. YOUNG,1 K. SWEENEY1 AND J. M. BEDFORD1 From the Cornell University Medical College, New York

SUMMARY The activity of the embryonic genome prior to the first cleavage has been assessed by studying the uptake of [3H]uridine, its phosphorylation and incorporation into RNA by mouse one-cell embryos. One-cell embryos incorporated [3H]uridine linearly into cold trichloracetic acid (TCA) insoluble material at a low level 1-9 h post fertilization. The incorporation of [3H]guanosine was also low but followed a biphasic curve which had a steeper slope at 1-3 h than during the period 4-9 h post fertilization. Unfertilized mouse ova incorporated very little [3H]uridine or [3H]guanosine into TCA insoluble material, and much of this was RNase insensitive. Dimethyl sulfoxide (DMSO) enhanced the uptake of [3H]thymidine and its incorporation into pronuclear DNA by one-cell embryos, but had no effect on the incorporation of [3H]uridine by them, or of [3H]uridine and [3H]guanosine by unfertilized ova. The uptake and incorporation of [3H]guanosine by one-cell embryos were enhanced by DSMO, but only during the period 1-3 h post fertilization. Sugar derivatives of UDP, and UMP, UDP, UTP, CMP, CDP and CTP have been identified in the soluble fraction obtained from mouse one-cell embryos incubated with [3H]uridine 1-3 h post fertilization. Very little of the [3H]uridine taken up by the embryos is present as [3H]UTP, or [3H]CTP; most is found as [3H]UMP or [3H]UDP or as the sugar 3 derivatives. Alkaline or ribonuclease (A, Tx and T2) hydrolysis of the H-labeled ethanol insoluble material precipitated from the lysate of one-cell embryos incubated with [3H]uridine 1-3 h post fertilization liberated radioactive cytidine and uridine-3'-phosphates. This demon- strates that [3H]uridine is incorporated into an internal position in RNA and suggests that RNA synthesis does occur in the one-cell embryo 1-3 h post fertilization. Since pronuclei of one-cell embryos incubated with [3H]uridine were not labeled it appears, however, that the RNA synthesized at the one-cell stage is not a product of the embryonic genome.

INTRODUCTION It is not yet clear whether activity of the embryonic genome is required at all stages of mammalian preimplantation development, and if not, when embryonic genes are first activated. The mouse preimplantation embryo does synthesize RNA by the two-cell stage of development, although the RNA has not been characterized (Knowland & Graham, 1972), but whether the embryonic genome is transcribed earlier, i.e. prior to the first cleavage, is not known. A low level of [3H]uridine is incorporated into TCA insoluble material by one-cell embryos

1 Authors' address: Reproductive Biology Unit, Departments of Obstetrics and Gynecology and Anatomy, Cornell University Medical College, 1300 York Avenue, New York, N.Y. 10021, U.S.A. 134 R. J. YOUNG, K. SWEENEY AND J. M. BEDFORD but attempts to isolate [3H]uridine-labeled RNA from them have been unsuccessful (Monesi & Salfi, 1967; Woodland & Graham, 1969; Monesi & Molinaro, 1971; Daentl & Epstein, 1971; Knowland & Graham, 1972; Graham, 1973). The one-cell embryo has also been reported to incorporate very little [3H]- guanosine, [32P]phosphate or [14C]carbonate into RNA (Woodland & Graham, 1969), and with the exception of one early autoradiographic study which reported occasional labeling of the pronuclei of one-cell embryos (Mintz, 1964) attempts to demonstrate RNA polymerase activity in the embryo have also been unsuccessful (Warner & Hearn, 1977; Moore, 1975). Thus, to date there has been no direct demonstration that RNA is synthesized by the one-cell embryo and this possibility rests only on the observation that inhibitors of RNA synthesis, actinomycin D and a-amanitin, inhibits cleavage of these embryos (Golbus, Calarco & Epstein, 1973). However, such inhibitors can affect metabolism in ways unrelated to the inhibition of RNA synthesis, and thus the effect of these on the first cleavage may not be due specifically to inhibition of RNA synthesis. While it is possible that the one-cell embryo is transcriptionally inactive, it is also possible that there may be difficulty in detecting a newly synthesized labeled RNA because of (a) non-entry of the labeled precursor (Woodland & Graham, 1969; Know- land & Graham, 1972; Graham, 1973), (b) the non-conversion of the labeled nucleoside to the triphosphate, (c) a level of RNA synthesis insufficient for its isolation and characterization by the technique of polyacrylamide gel electrophoresis (Knowland & Graham, 1972; Graham, 1973), or (d) discontinuous synthesis from the time of fertilization until the first cleavage. These possibilities have been explored in this communication which shows that uridine can enter the mouse one-cell embryo, is phosphorylated, and is incorporated at a low level into newly synthesized RNA.

MATERIALS AND METHODS Collection of ova Virgin random-bred Swiss female mice 7-12 weeks old were superovulated by intraperitoneal injection with 7-5-10 units each of pregnant mare's serum (Equinex, Ayerst) and 46-48 h later with human chorionic gonadotropin (HCG) (Pregnyl, Organon). Unfertilized eggs were collected from females sacrificed 13-16 h post HCG. To obtain fertilized eggs, a female was placed with a male (Balb CJ x C57 BL/6J) immediately after injection with HCG, and checked for the presence of a vaginal plug early next morning. Mated females were sacrificed commencing 16-16-5 h post HCG. Cumulus cells were removed from ova by incubation with hyaluronidase (Sigma, type VI, 150 units/ml) in Whitten's medium (Whitten, 1971) containing 10 mg/ml bovine serum albumin (BSA) (Miles) for 10 min followed by three washes with medium. The collection Undine incorporation in mouse embryo 135 of ova was carried out at 37 °C using medium equilibrated with a gas mixture of 5 % O2, 5 % CO2J 90 % N2 and overlayered with silicon oil (Dow Corning 200 Dielectric Fluid).

Culture and labeling of ova Eggs collected from females at intervals of 1 h (unmated) and 2 h (mated) were distributed into two groups of 200-400 each, and incubated in 100/tl drops of Whitten's medium under oil at 37 °C in an atmosphere of 5 % O2, 5 % CO2 and 90 % N2. The medium for one group of eggs contained 1 % DMSO. For labeling of eggs, tritiated nucleosides were present at 100-500 /*Ci/ ml of medium. After incubation with label for 2 h the eggs were washed with three to five changes of medium containing 0-1 mg/ml of unlabeled nucleoside, and the eggs in each group transferred in batches of 25-60 to 25 /i\ of lysing buffer (0-1 M Tris-HCl, pH 7-5, 1 % sodium dodecyl sulfate (SDS), 10 jug yeast RNA (Sigma, type VI)) and frozen. Approximately 30 % of the eggs collected from mated females had pronuclei 16-17 h post HCG, increasing to about 90 % at 23-24 h post HCG (Luthardt 6 Donahue, 1973). The percentage of pronuclear eggs was determined at the first time point and after a further 4-6 h to provide a guide to the extent of fertilization and development of the fertilized egg. The maturation of mouse follicular oocytes in vitro was carried out as described by Cross & Brinster (1970) except that the prior injection of PMS was omitted. Oocytes were cultured in the absence of cumulus cells in simple serum medium with or without 1 % DMSO as required for the experiment. For labeling experiments, [5, 6-3H]uridine at 500 jLtCi/mi was present. At intervals after being placed in culture, groups of eggs were removed and washed as above. Epididymal spermatozoa were used for in vitro fertilization experiments (Hoppe & Pitts, 1973). Fertilization rates of 70-80 % were achieved when eggs were collected at 13 h post HCG, with not more than 20-30 eggs per dish. When egg numbers were increased (up to 80/dish), the fertilization rate fell to 5-20 %. DMSO is soluble in water, but is not miscible with silicon dielectric fluid (2 /i\ does not dissolve in 5 ml of the fluid), and is therefore not lost from the incubation medium. Tritiated nucleosides, [5-3H]uridine (specific activity 25-29 Ci/mmole), [5, 6-3H]uridine (specific activity 45-50 Ci/mmole), [8-3H]guanosine (specific activity 9-11 Ci/mmole), [methyl-3H]thymidine (specific activity 40-60 Ci/ mmole) and [5, 6-3H]uridine-5'-triphosphate (specific activity 35-50 Ci/mmole) were obtained from New England Nuclear or Amersham/Searle.

Measurement of nucleoside incorporation Radioactivity was measured after lysis of ova by repeated freeze-thawing by one of two methods. For the first (a) cold 50 % TCA was added to the lysate 136 R. J. YOUNG, K. SWEENEY AND J. M. BEDFORD to a final concentration of 10 %, and after 1 h at 0 °C the mixture was centri- fuged at 10000 g in the cold. The pellet was washed three times with cold 5 % TCA, the supernatants combined, and the TCA soluble radioactivity determined by counting the supernatant in a liquid scintillation spectrometer after adding a toluene-based scintillation fluid (4/g Omnifluor/liter toluene) (New England Nuclear) containing BBS3 (Beckman). The TCA insoluble pellet was dissolved in 0-1 M ammonium hydroxide before measurement of the radioactivity. The second method (b) is similar to that previously described (Johnson & Young, 1969). The lysate was quantitatively transferred onto 25 mm discs of Whatman No. 1 filter paper, dried, and the radioactivity on the discs measured. The paper discs were then washed in toluene, and then in cold 5 % TCA, cold 95 % ethanol, and cold ether, dried and counted. The first measurement gives an estimate of the total nucleoside uptake, and the second, the amount of TCA insoluble radioactivity. Backgrounds were determined by adding an aliquant of the medium used for the final wash to the lysing buffer, and carrying this through the precipitation and washing procedure. The second method (b) was more convenient for the assay of many batches of embryos but the efficiency of counting was lower because of self absorption. Larger numbers of embryos or a higher level of incorporation was required when the time course of incorporation was assayed by this method. Samples were counted for a time sufficient to accumulate a minimum of 1000 background counts. Background varied between 15 and 20cpm. Each experimental point represents the average from two or three batches of 25-60 eggs, and most kinetic experiments were carried out three times. Labeling times given are the hours after HCG at which the eggs were first placed into culture. Autoradiography (Luthardt & Donahue, 1973) was carried out using Kodak NTB2 liquid emulsion. Sample slides were checked at 10 days and then at 3-day intervals.

Isolation of labeled material Incubation of up to 700 embryos was carried out with gentle shaking on a reciprocal shaker for 2h (17-19 h post HCG) in 100 jil drops of Whitten's medium containing [5, 6-3H]uridine at 500/6Ci/ml. Embryos were then washed 4-6 times in medium containing 0-1 mg/ml of uridine and then transferred to lysing buffer. The embryos were lysed by repeated freeze-thawing and insoluble 3H-labeled material isolated as described (Young, 1977). This method gave good recovery of insoluble material, but some soluble nucleotides were also present as a contaminant. Three cycles of precipitation usually reduced con- tamination of the insoluble 3H-labeled material by soluble nucleotides, mainly UTP and UDP, to 0-1-1 % of the soluble fraction. The 3H-labeled precipitate was suspended in 0-1 M Tris-HCl, pH 7-5, incubated at 37 °C with pronase (self-digested, nuclease free (Calbiochem)) for 2 h, and RNA isolated as Uridine incorporation in mouse embryo 137 described by Perry, La Torre, Kelley & Greenberg (1972). Controls in which E. coli rRNA was present in the lysate showed that this procedure did not result in degradation of RNA. For isolation of soluble 3H-labeled material embryos were suspended in buffer containing 10-15 /.ig UTP but no SDS. After repeated freeze-thawing the insoluble material was precipitated as described above, collected by centri- fugation and washed with 70 % aqueous ethanol. The precipitate was dispersed, resuspended in water and re-precipitated with 2 vol of ethanol. This procedure was repeated three to four times. The supernatant and 70 % ethanol washes from the precipitations were combined, concentrated and the composition of the soluble 3H-labeled material analyzed by chromatography on polyethylene- imine impregnated (PEI) cellulose thin layer sheets. Hydrolysis by KOH of the insoluble 3H-labeled material obtained by this procedure from three batches of embryos (total 2500), followed by paper electrophoresis of the hydrolysate at pH 3-5, showed that little or no UDP or UTP was present in the insoluble fraction. Thus this procedure does not result in a loss of 3H-labeled material from the soluble to the insoluble fraction. Alkaline and enzymic digestion Alkaline hydrolysis was carried out in 0-3 M-KOH at 37 °C for 16-20 h. The hydrolysate was desalted with perchloric acid before chromatography. Embryo lysate not extracted with phenol-chloroform and carrier yeast tRNA (7 /tg) was digested with ribonucleases Tx (15 units), T2 (10 units) (Sigma) and ribonuclease A (5 jug) (Worthington) in 005 M sodium acetate, pH 5 and 0-003 M-EDTA for 8 h at 37 °C. The pH was adjusted to 7-5 and incubation continued for 2 h with pronase (200 /tg) before chromatography. Thin-layer chromatography and paper electrophoresis Thin-layer chromatography was carried out on PEI-cellulose sheets (Baker) as described (Young, 1977). Solvent systems were: step formate and step LiCl systems (Randerath & Randerath, 1964); solvent A, 0-15 M sodium borate- 0-5 M boric acid in 25 % ethylene glycol; B, 0-9 M acetic acid - 0-1 M LiCl; c, 1 M LiCl; and D 1 M acetic acid. The PEI-cellulose sheets were washed with anhydrous methanol after spotting the sample and markers and also after chromatography in the first dimension. A spot of equal area adjacent to the markers was used as background. Paper electrophoresis was carried out in citrate buffer as previously described (Young & Fraenkel-Conrat, 1971).

RESULTS Nucleoside incorporation by one-cell mouse embryos and unfertilized mouse ova There was a low level of incorporation of [3H]thymidine by one-cell embryos into TCA insoluble material throughout the period 17-25 h post HCG 138 R. J. YOUNG, K. SWEENEY AND J. M. BEDFORD

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21 23 25 27 Time post HCG (h) Fig. 1. Effect of DMSO on uptake and incorporation of [3H]thymidine by one-cell mouse embryos. (A) TCA soluble radioactivity; O, 1% DMSO; •, no DMSO. (B) TCA insoluble radioactivity; 0,1 % DMSO; •, no DMSO. Each time point is the time post HCG when the embryos were placed in the culture medium for a 1 h incubation. Radioactivity was measured by method (b). One experiment is shown and each experimental point is the average value from 2 or 3 batches of embryos. The nucleoside was present at a concentration of 500 /tCi/ml.

(Fig. IB). However, at about 21 h there was an increase in the level to reach a maximum at 23-25 h before returning to the basal level at about 26 h post HCG or about 4-6 h before the first cleavage division. The TCA insoluble radioactivity was reduced by 70-80 % after incubation of embryo lysate with DNase showing that DNA is synthesized during this period. Because of asyn- chrony of fertilization, pronuclear DNA synthesis will commence at different times in different embryos and [3H]thymidine incorporated into TCA insoluble material in the period 17-20 h post HCG may also represent pronuclear DNA synthesis. The latter has also been studied by autoradiographic and cytophoto- metric methods (Luthardt & Donahue, 1973; Siracusa, Coletta & Monesi, 1975; Ambramczuk & Sawicki, 1975), and the results are in excellent agreement Uridine incorporation m mouse embryo 139

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00 1 1 1 16 17 18 19 20 Time post HCG Fig. 2. Uptake and incorporation of tritiated nucleosides by one-cell mouse embryos in the presence or absence of DMSO. (A) [5-3H]uridine; •, TCA soluble, no DMSO; O, TCA soluble, 1 % DMSO; •, TCA insoluble, no DMSO; •, TCA insoluble, 1% DMSO. (B) [3H]guanosine; •, TCA soluble, no DMSO; O, TCA soluble, 1% DMSO; •, TCA insoluble, no DMSO; D, TCA insoluble, 1% DMSO. Each time point is the time post HCG when the embryos were placed in the culture medium for 2 h. The figure shows an experiment in which the radioactivity was measured by method (a). Each point is the average value from two-three batches of embryos. The concentration of the nucleosides was 500 /tCi/ml. with those of the present study showing that the present method is sufficiently sensitive to measure low levels of incorporation of label. [3H]Uridine was found to be incorporated at a low level into TCA insoluble material in agreement with the studies of other workers (Monesi & Salfi, 1967; Monesi & Molinaro, 1971; Knowland & Graham, 1972). The incorporation of [3H]uridine remained at this low level throughout the period 16-24 h post HCG (Fig. 2 A), but in contrast to the behaviour of [3H]uridine, [3H]guanosine incorporation was not linear being higher 16-18 h post HCG than during later stages of development, i.e. the kinetic curve for guanosine is biphasic (Fig. 2B). These results suggest that [3H]uridine is incorporated into macromolecules at a 140 R. J. YOUNG, K. SWEENEY AND J. M. BEDFORD

Table 1. Incorporation of [zH]uridine by maturing mouse follicular oocytes

Total radioactivity TCA insoluble radioactivity (cpm/egg) (cpm/egg) Time in , A > , -* > culture (h) -DMSO +1 % DMSO -DMSO 1-3 12 16 1-9 3-5 2-2 11 17 2-6 7-4 4-5 14 66 3-3 11-2 240 78 83 25-0 310

constant low rate throughout the development of the one-cell embryo, whereas more [3H]guanosine is incorporated 1-3 h after fertilization than at later periods during development of the one-cell embryo. The estimate of the time of fertilization (15-16 h post HCG) after natural mating as in the present experiments is imprecise, and in vitro fertilization (Hoppe & Pitts, 1973) was attempted in order to determine more accurately the commencement of [3H]uridine and [3H]guanosine incorporation. However, the percentage of eggs fertilized was found to vary inversely with the number of eggs present per drop of medium and in vitro fertilization could not provide sufficient fertilized eggs to enable a kinetic study of nucleoside incorporation. Unfertilized ova incorporated very little [3H]uridine and [3H]guanosine into cold TCA-insoluble material during the period 1-6 h post ovulation. More than 80 % of the [3H]guanosine labeled and 60-70 % of [3H]uridine-labeled material was insensitive to RNase A, showing that no [3H]guanosine and little [3H]- uridine was incorporated into RNA; the latter incorporation is most probably turnover incorporation into the -CCA end of tRNA. This result is in agreement with the expectation that RNA synthesis should not occur in the ovulated ovum where the chromosomes are in condensed state. The identity of the cold TCA- insoluble 3H-labeled material is unknown.

Effect of DMSO on nucleoside incorporation The low level of [3H]nucleoside incorporation by the one-cell embryo has been postulated to be due to its restricted entry (Woodland & Graham, 1969; Knowland & Graham, 1972; Graham, 1973). Since DMSO can increase the permeability of cellular membranes to many solutes its effect on the incorporation of nucleosides by one-cell embryos was studied. Preliminary experiments showed that 1 % DMSO did not inhibit the cleavage of fertilized mouse ova. Fig. 1 shows that 1 % DMSO enhanced both the uptake and incorporation of [3H]thymidine by one-cell embryos. This was observed first at about 21 h, the time of commencement of pronuclear DNA synthesis (Fig. 1B) with the maximum enhancement occurring during the time of maximum pronuclear DNA synthesis at 23-25 h post HCG. Its enhancement Uridine incorporation in mouse embryo 141 of [3H]thymidine incorporation during the pronuclear DNA synthetic period is low (assayed by method b) but since the percentage increment in uptake (Fig. 1 A) is approximately equal to the percentage increment in incorporation (Fig. 1 B), the incorporation of [3H]thymidine is most probably maximal under the conditions of incubation. Thus, although low, the enhancement by 1 % DMSO of [3H]thymidine incorporation into pronuclear DNA is real, and was reproducible. The effect of 1 % DMSO on the incorporation of [3H]uridine by mouse follicular oocytes maturing in vitro was also studied since [3H]uridine is incorporated by follicular oocytes after 2-6 h in culture (Bloom & Mukherjee, 1972). The data presented in Table 1 show that [3H]uridine uptake and incorporation by follicular oocytes is in fact enhanced by 1 % DMSO. Therefore in the maturing follicular oocyte and the one-cell embryo, where the synthesis of RNA and DNA respectively has been demonstrated by other methods, 1 % DMSO has been found to enhance the uptake and incorporation of nucleoside precursor. By contrast, the uptake and incorporation of [3H]uridine by one-cell embryos (Fig. 2 A) was not affected by DMSO over the period 16-24 h post HCG. However, DMSO enhanced (Fig. 2B) the uptake and incorporation of [3H]- guanosine 16-18 h post HCG, a period in which the nucleoside is incorporated into macromolecules, but at 18-24 h post HCG when less [3H]guanosine was incorporated, DMSO had no effect on this. Thus the curve of [3H]guanosine incorporation by the one-cell embryo 16-24h post HCG is biphasic, whether DMSO is present or absent. The uptake and incorporation of [3H]uridine and [3H]guanosine by ovulated unfertilized mouse ova were unaffected by 1 % DMSO. These results indicate that while DMSO can enhance the uptake and incorporation of nucleosides into macromolecules by the ovum, their transport into the ovum appears to depend on their utilization for synthesis. The low level of [3H]uridine incorporation into macromolecules by the one-cell embryo is probably therefore not due to the inability of the nucleoside to enter the embryo.

Composition of the labeled soluble fraction The failure of nucleoside precursor to be converted to the triphosphate seems a possible reason for the low level of incorporation of [3H]uridine into a TCA insoluble product by the one-cell embryo. Examination of the soluble [3H]uridine-labeled material present in the lysate of embryos 17-19 h post fertilization by paper electrophoresis at pH 3-5 and by thin layer chromatography with the step formate system showed that UMP, UDP and UTP were present in the soluble fraction, but also that the composition of the soluble fraction was complex. However, a two-dimensional thin layer chromatographic system (Randerath & Randerath, 1964) resolved the 10 EMB 44 142 R. J. YOUNG, K. SWEENEY AND J. M. BEDFORD

Table 2. Undine nucleotides present in one-cell embryos 1-3 h post fertilization*

Nucleotide Percentagef UMP 42-7 UDP 29-9 UTP 30 CMP 1-5 CDP 0-83 CTP 0-42 UDPG 16-3 UDPGAJ 0-66 CDPG 0-77 X 3-9 * Components resolved by two-dimensional chromatography on PEI-cellulose. First dimension: step LiCl; second dimension: step formate (Randerath & Randerath, 1964). t Average value from four experiments. % Uridine diphosphate N-acetylglucosamine.

3H-labeled material present in the soluble fraction into nine radioactive components which co-migrated with added markers. A tenth radioactive component, that migrated slightly ahead of both uridine diphosphate glucose (UDPG) and cytidine diphosphate glucose (CDPG) in the step LiCl system and had the same mobility as CDPG in the step formate system, was also detected in the two- dimensional chromatogram. It is clear that some UTP, the immediate precursor of RNA, is present in the one-cell embryo but that most of the [3H]uridine is present as the UMP or UDP or as nucleoside diphosphate sugars (Table 2). This result shows that [3H]uridine is able to enter the mouse one-cell embryo, and upon entry is made available for RNA synthesis by conversion to the triphosphate. The difficulty in detecting [3H]uridine incorporation into RNA is probably not due to the unavailability of the nucleoside or its triphosphate. Paper electrophoresis and paper chromatography showed that cytidine was not present as a contaminant in the [5, 6-3H]uridine precursor. The presence of small amounts of CTP, CDP and CMP in the soluble fraction therefore demon- strates that pyrimidine interconversion can occur in the one-cell embryo, although this interconversion apparently does not take place at the later stages of development (Woodland & Graham, 1969).

The nature of the labeled insoluble fraction Even when 500-2000 one-cell embryos were used in previous studies, labeled RNA could not be resolved by gel electrophoresis although TCA insoluble material was present (Knowland & Graham, 1972; Graham, 1973); this material however was not tested for its stability toward RNase or alkali by Undine incorporation in mouse embryo 143 C5

Solvent B then Solvent C UDPG c

Solvent A Fig. 3. Two-dimensional thin layer chromatogram of an alkaline hydrolysate of 3H- labeled RNA isolated by phenol-chloroform extraction of one-cell embryos that had been incubated with [5, 6-3H]uridine 1-3 h post fertilization. First dimension: solvent A (right to left). Second dimension: solvent B, then dried and washed with anhydrous methanol and developed further in solvent C until front was 1 cm below Up spot (bottom to top). these or other workers (Monesi & Salfi, 1967; Daentl & Epstein, 1971). In- cubation of the lysate of embryos labeled with [5, 6- 3H] uridine or [3H]guanosine overnight with RNase A or 0-3 M-KOH reduced the amount of TCA insoluble radioactivity present, but only by 40-60 %. Thus the labels are incorporated into RNA as well as into material which is not RNA. An attempt to isolate RNA by phenol-chloroform extraction of the pronase-digested lysate of embryos which had been incubated with [3H]guanosine was unsuccessful; most (85-95 %) of the radioactivity was present in the aqueous layer, but very little was precipitated from solution by ethanol. Therefore very little of the nucleosides uridine or guanosine are incorporated into RNA by the one-cell embryo and the failure of gel electrophoresis (Graham, 1973; Knowland & Graham, 1972) to detect and resolve labeled RNA was most likely due to the small quantity of material available. Since pyrimidine interconversion occurs in the one-cell embryo (see above) the 3H-labeled TCA insoluble material present in embryos incubated with [3H]uridine may represent turnover incorporation of [3H]cytidine into the -CCA terminus of tRNA rather than [3H]uridine incorporation into newly synthesized RNA. Chemical degradation of the labeled material was studied to determine whether both UTP and CTP are incorporated into RNA. Alkali or 144 R. J. YOUNG, K. SWEENEY AND J. M. BEDFORD

a mixture of the enzymes ribonuclease A, Tx and T2 will degrade RNA into a mixture of nucleoside-3'-phosphates. Since these nucleotides do not occur naturally, their presence in such digests would prove the existence of RNA. Using this approach, 3H-labeled material isolated by ethanol precipitation from one-cell embryos incubated 17-19 h post HCG with [3H]uridine was digested with pronase and the digest extracted with phenol-chloroform. The labeled RNA in the aqueous layer was hydrolyzed with alkali and the hydrolysate examined by two-dimensional thin layer chromatography (Fig. 3). Preliminary experiments showed that small quantities of uridine-5'-phosphate were present in the insoluble 3H-labeled material and UMP (pU) as well as UTP, UDP were found to be radioactive. In addition the pyrimidine-3'- phosphates, Up and Cp, products of alkaline hydrolysis, were radioactive, but CMP (pC), which is not a product of alkaline hydrolysis and was not present as a contaminant, was not radioactive. The same result was obtained if the ethanol insoluble 3H-labeled material was digested with alkali or with a mixture of ribonucleases A, Tx and T2 without prior extraction with phenol-chloroform. The separation of Cp, pC, Up and pU from each other and from contaminating UDP, UTP and uridine diphosphate sugars could also be effected by using step LiCl in the first dimension followed in the second dimension by solvent D until the front was 9 cm from the origin, then with further development without intermediate drying in solvent B until the front reached the top of the thin layer sheet. In this system radioactivity was also associated with Cp, Up and pU but not with pC. In three experiments using between 3000 and 3600 embryos per experiment, the amount of radioactivity found in RNA, measured as Cp and Up, averaged 383 cpm. Thus both uridine and cytidine are incorporated at a low level into internal positions in RNA by the one-cell embryo, and terminal addition of [3H]cytidine is not the sole contributor of 3H-label to the 3H- labeled RNA.

Autoradiography Autoradiographs of embryos incubated with [3H]thymidine showed distinct labeling of both pronuclei. When [3H]uridine or [3H]UTP was used neither the pronuclei nor the ooplasm was labeled even after an exposure period of 3-4 weeks.

DISCUSSION Examination of nucleoside uptake by mouse preimplantation embryos has indicated that uridine and thymidine share the same or a similar transport system (Daentl & Epstein, 1973). Since thymidine (Fig. 1) as well as [3H]- guanosine (Fig. 2B), [3H]adenosine and [32P]phosphate are taken up by the one-cell embryo (Young, 1976) and [3H]uridine is able to enter the maturing oocyte it is unlikely that its cell membrane is impermeable to [3H]uridine. The present results (Fig. 2 A) and those of Warner & Hearn (1977) show that Undine incorporation in mouse embryo 145 [3H]-uridine is taken up by the one-cell embryo. Difficulties in labeling its RNA is therefore unlikely to be due to failure of [3H]uridine to enter the embryo. Thus the low level of incorporation of [3H]guanosine after 18 h post HCG (Fig. 2B), and the biphasic nature of the incorporation curve may explain the failure of early workers to detect incorporation of this nucleoside by the one- cell embryo (Woodland & Graham, 1969). Since the chromosomes of unfertilized ova are at the metaphase II stage and are condensed, RNA synthesis would be unexpected and indeed DMSO does not enhance the uptake of [3H]uridine or [3H]guanosine. This solvent does enhance the uptake of [3H]thymidine (Fig. 1A) and [3H]guanosine (Fig. 2B) by the one-cell embryo and [3H]uridine by the maturing oocyte (Table 1) when the embryo and oocyte are active in the synthesis of nucleic acids. It is possible that the entry of nucleosides and the ability of DMSO to enhance the entry of nucleosides into mouse embryos and ova are related to the level of utilization of the nucleoside for nucleic acid synthesis. Therefore the inability of DMSO to enhance the uptake or incorporation of [3H]uridine by one-cell embryos (Fig. 2 A) may mean that the level of its RNA synthesis is low. This conclusion is supported by the recent observation that the amount of [3H]uridine taken up by the one-cell embryo is in the same order of magnitude as that taken up by the two-cell stage (Warner & Hearn, 1977). The latter is active in RNA synthesis whereas the former is not (Knowland & Graham, 1972). The conversion of [3H]uridine to [3H]uridine phosphates has been noted in two- and eight-cell embryos, and in the blastocyst (Woodland & Graham, 1969; Daentl & Epstein, 1971). The present results show that [3H]uridine is taken up and phosphorylated by the one-cell embryo as early as 3 h post fertilization, but that most is found as UMP and UDP and as sugar derivatives of UDP rather than as UTP (Table 2). Sugar derivatives of UDP have not previously been found in mouse embryos, but can be anticipated there in view of the increase in glycogen during development from the one-cell to the two-cell stage (Stern & Biggers, 1968; Ozias & Stern, 1973). The large amount of UDP- sugar derivatives present in the soluble uridine nucleotide pool indicates that the low percentage of UTP in the pool is not due to low uridylate kinase activity. The percentage of [3H]uridine found as UTP at the two-cell and later stages of development has been reported to vary between 40 % and 70 % and these stages are also active in RNA synthesis (Daentl & Epstein, 1971; Ellem & Gwatkin, 1968; Woodland & Graham, 1969; Monesi & Salfi, 1967; Piko, 1970; Tasca & Hillman, 1970; Monesi & Molinaro, 1971; Knowland & Graham, 1972). In contrast the present work shows that only 3 % of the [3H]- uridine taken up by the one-cell embryo 1-3 h post fertilization is present as [3H]UTP; thus there is little UTP available for RNA synthesis at the one-cell stage and consistent with this only a small amount of TCA-insoluble 3H-labeled material has been isolated from such embryos (Knowland & Graham, 1972; Graham, 1973). The difficulty in detecting newly labeled RNA in the one-cell 146 R. J. YOUNG, K. SWEENEY AND J. M. BEDFORD embryo is therefore most probably due to the very low level of RNA synthesis rather than to restricted entry of nucleoside precursors or to inactivity of the kinases required to convert the nucleoside to the triphosphate. Much of the UTP appears to be utilized for polysaccharide rather than for RNA synthesis, since the percentage of UDP-sugar derivates is high. The presence of radioactive Up and Cp in alkaline and enzymic digests of one-cell embryos incubated with [3H]uridine up to 4 h after fertilization or in their 3H-labeled material shows that the 3H-labeled material is RNA and that both Cp and Up are present in internal positions in the RNA. The one-cell embryo is active in protein synthesis (Epstein & Smith, 1973; Van Blerkom & Brockway, 1975) and incorporation of CTP into the 3'-terminal-CCA segment of tRNA would be expected. At least part of [3H]Cp found in the alkaline and enzymic digest would be derived from this source. Uridine is not known to have a similar turnover role, and unless [3H]uridine incorporation is simply a terminal addition to existing RNAs, the finding of Up means that RNA is synthesized by the one-cell embryo albeit at a low level. Furthermore, this low level of incorporation occurs continuously soon (1-3 h) after fertilization until after the onset of pronuclear DNA synthesis (Fig. 2 A) suggesting that the RNAs made have similar functions and are synthesized continuously after activation at fertilization. On the other hand the biphasic nature of the [3H]guanosine incorporation curve (Fig. 2B) indicates that the [3H]guanosine-labeled macromolecules synthesized soon after fertilization are either different from those appearing at a later stage, or simply that the macromolecules are the same but the rate of synthesis is higher soon after fertilization. For example, after fertilization [3H]guanosine may be incorporated into pre-existing mRNA as the 7- methylguanosine capped structure, thus converting the mRNA into an active form (Mulhukrishnan, Both, Furuichi & Shatkin, 1975; Both, Banerjee & Shatkin, 1975a; Both, Furuichi, Mulhukrishnan & Shatkin, 19756; Young, 1977). In an autoradiographic study of [3H]uridine incorporation by mouse one-cell embryos, Mintz (1964) reported that pronuclei of some embryos but not the cytoplasm were labeled. A later autoradiographic study employing a technique which enables [3H]UTP to enter the embryo failed to confirm pronuclear labeling although one polar body was frequently found to be labeled (Moore, 1975). We also have not observed labeling of pronuclei in one-cell embryos incubated with [3H]uridine. Thus, the RNA synthesized soon after fertilization may not be a transcript of the embryonic genome; it is also unlikely that the RNA is a product of the mitochondrial genome since mitochondria of the embryo appear not to be active in RNA synthesis before the eight-cell stage (Piko, 1975; Piko & Chase, 1973) and the cytoplasm of the embryo was not labeled. It is possible therefore that the [3H]uridine-labeled RNA present in the one-cell embryo is synthesized by a polar body. If this is the case it is unlikely that this RNA plays a role in the first cleavage of the fertilized ovum, and development of the embryo to the two-cell stage does not require Uridine incorporation in mouse embryo 147 pronuclear RNA synthesis. The possibility cannot be excluded that the embryonic genome is active and that the level of RNA synthesized is too low to be detected consistently by autoradiography. This putative RNA is unlikely to be mRN A but may have a regulatory role or may serve as a primer for DNA repli- cation. Thus, although a low level of RNA synthesis is shown here to occur in the one-cell embryo, this may not be a transcript of the embryonic genome and development of the mouse embryo up to the two-cell stage may depend on a store of maternal RNA.

Part of this work was carried out in the Laboratory of Reproductive Physiology, University of Pennsylvania. The hospitality and encouragement extended by Dr R. L. Brinster and discussions with Dr P. Cross and Dr G. B. Stull are gratefully acknowledged. This investiga- tion was supported by The Rockefeller Foundation and The National Institutes of Health.

REFERENCES ABRAMCZUK, J. & SAWICKI, W. (1975). Pronuclear synthesis of DNA in fertilized and parthenogentically activated mouse eggs. Expl Cell Res. 92, 361-372. BLOOM, A. M. & MUKHERJEE, B. B. (1972). RNA synthesis in maturing mouse oocytes. Expl Cell Res. 74, 577-582. BOTH, G. W., BANERJEE, A. K. & SHATKIN, A. J. (1975a). Methylation-dependent translation of viral messenger RNA's in vitro. Proc. natn. Acad. Sci. U.S.A. 72, 1189-1193. BOTH, G. W., FURUICHI, Y., MULHUKRISHNAN, S. & SHATKIN, A. J. (19756). Ribosome binding to reovirus mRNA in protein synthesis required 5'-terminal 7-methylguanosine. Cell 6, 185-195. CROSS, P. C. & BRINSTER, R. L. (1970). In vitro development of mouse oocytes. Biol. Reprod. 3, 298-307. DAENTL, D. L. & EPSTEIN, C. J. (197.1). Developmental interrelationships of uridine uptake nucleotide formation and incorporation into RNA by early mammalian embryos. Devi Biol. 24, 428-442. DAENTL, D. L. & EPSTEIN, C. J. (1973). Uridine transport by mouse blastocysts. Devi Biol. 31, 316-322. ELLEM, K. A. O. & GWATKIN, R. B. L. (1968). Patterns of nucleic acid synthesis in the early mouse embryo. Devi Biol. 18, 311-330. EPSTEIN, C. J. & SMITH, S. A. (1973). Amino acid uptake and protein synthesis in preimplantation mouse embryos. Devi Biol. 33, 171-184. GOLBUS, M. S., CALARCO, P. G. & EPSTEIN, C. J. (1973). The effects of inhibitors of RNA synthesis (a-amanitin and actinomycin D) on preimplantation mouse embryogenesis. J. exp. Zool. 186, 207-216. GRAHAM, C. F. (1973). Nucleic acid metabolism during early mammalian development. In The Regulation of Mammalian Reproduction (ed. S. Segal, R. Crozier, P. A. Corfman & P. G. Condliffe), pp. 286-301. Springfield, 111.: Charles C. Thomas. HOPPE, P. C. & PITTS, S. (1973). Fertilization in vitro and development of mouse ova. Biol. Reprod. 8, 420-426. JOHNSON, M. S. & YOUNG, R. J. (1969). Fractionation on benzyolated DEAE-cellulose of tRNA from tobacco mosaic virus-infected tobacco leaves. Virology 38, 607-613. KNOWLAND, J. & GRAHAM, C. (1972). RNA synthesis at the two-cell stage of mouse development. /. Embryol. exp. Morph. 27, 167-176. LUTHARDT, F. W. & DONAHUE, R. P. (1973). Pronuclear DNA synthesis in mouse eggs. Expl Celt'Res. 82, 143-151. MINTZ, B. (1964). Synthetic processes and early development in the mammalian egg. /. exp. Zool. 157, 85-100. 148 R. J. YOUNG, K. SWEENEY AND J. M. BEDFORD MONESI, V. & MOLINARO, M. (1971). Macromolecular synthesis and effect of metabolic inhibitors during preimplantation development in the mouse. Schering Symposium on Intrinsic and Extrinsic Factors in Early Mammalian Development (ed. G. Raspe). Oxford: Pergamon Press. Adv. Biosciences 6, 101-117. MONESI, Y. & SALFI, V. (1967). Macromolecular synthesis during early development in the mouse embryo. Expl Cell Res. 46, 632-635. MOORE, G. P. M. (1975). The RN A polymerase activity of the preimplantation mouse embryo. /. Embryol. exp. Morph. 34, 291-298. MULHUKRISHNAN, S., BOTH, G. W., FURUICHI, Y. & SHATKIN, A. J. (1975). 5'-Terminal 7-methylguanosine in eukaryotic mRNA is required for translation. Nature, Lond. 255, 33-37. OZIAS, C. B. & STERN, S. (1973). Glycogen levels of preimplantation mouse embryos develop- ing in vitro. Biol. Reprod. 8, 467-472. PERRY, R. P., LA TORRE, J., KELLEY, D. E. & GREENBERG, J. R. (1972). On the lability of Poly (A) sequences during extraction of messenger RNA from polysomes. Biochim. Biophys. Acta 161, 220-226. PIKO, L. (1970). Synthesis of macromolecules in early mouse embryos cultured in vitro: RNA, DNA and a polysaccharide component. Devi Biol. 21, 257-279. PIKO, L. (1975). Expression of mitochondrial and nuclear genes during early development. In The Early Development of Mammals (ed. M. Balls & A. E. Wild), pp. 167-187. Cambridge: Cambridge University Press. PIKO, L. & CHASE, D. G. (1973). Role of the mitochondrial genome during early development in mice. Effects of ethidium bromide and chloramphenicol. /. Cell Biol. 58, 357-378. RANDERATH, K. & RANDERATH, E. (1964). Resolution of complex nucleotide mixtures by two-dimensional anion exchange thin layer chromatography. /. Chromat. 16, 126-129. SIRACUSA, G., COLETTA, M. & MONESI, V. (1975). Duplication of DNA during the first cell cycle in the mouse embryo. /. Reprod. Fert. 42, 395-398. STERN, S. & BIGGERS, J. D. (1968). Enzymatic estimation of glycogen in the cleaving mouse embryo. J. exp. Zool. 168, 61-66. TASCA, R. J. & HILLMAN, N. (1970). Effects of actinomycin D and cyclohexamide on RNA and protein synthesis in cleavage stage mouse embryos. Nature, Lond. 225, 1022-1025. VAN BLERKOM, J. & BROCKWAY, G. O. (1975). Qualitative patterns of protein synthesis in the preimplantation mouse embryo. Devi Biol. 44, 148-157. WARNER, C. M. & HEARN, T. F. (1977). The effect of a-amanitin on nucleic acid synthesis in preimplantation mouse embryos. Differentiation 7, 89-97. WHITTEN, W. K. (1971). Nutrient requirements for the culture of preimplantation embryos in vitro. Schering Symposium on Intrinsic and Extrinsic Factors in Early Mammalian Development (ed. G. Raspe). Oxford: Pergamon Press. Adv. Biosciences 6, 129-140. WOODLAND, H. R. & GRAHAM, C. F. (1969). RNA synthesis during early development of the mouse. Nature, Lond. Ill, 327-332. YOUNG, R. J. & FRAENKEL-CONRAT, H. (1971). The 3'-linked terminus of QB RNA. Biochim. Biophys. Acta 228, 446-455. YOUNG, R. J. (1976) Incorporation of nucleic acid precursors by unfertilized and fertilized mouse eggs prior to cleavage. Fed. Proc. 35, 1690. YOUNG, R. J. (1977). Appearance of 7-methylguanosine-5'-phosphate in the RNA of mouse 1-cell embryos three hours after fertilization. Biochem. Biophys. Res. Comm'.16, 32-39.

{Received 11 August 1977, revised 26 October 1977)