MOLECULAR REPRODUCTION AND DEVELOPMENT 31:182-188 (1992)

Expression of Ribosomal Protein Genes in Mouse Oocytes and Early Embryos KENT D. TAYLOR AND LAJOS PIKO Developmental Biology Laboratory, VA Medical Center, Sepulveda, California

maternal program is replaced by embryonic genetic control during the two-cell stage, when the zygote nuclei become fully transcriptionally active (Clegg and Piko, 1982) and the bulk of the maternal mRNA is degraded (Flach et al., 1982; Piko and Clegg, 1982). The number of ribosomes per embryo rises rapidly during subsequent cleavage, to about 10’ ribosomes in the eight-cell embryo and to about 2.5 x lo8 ribosomes in the blastocyst. This dramatic increase in ribosomal content between the two-cell and blastocyst stages is attributable entirely to the production of new ribosomes by the embryo (Piko and Clegg, 1982). The need for the early initiation of ribosome biosynthesis in the mouse embryo is the consequence of the relative sparsity of ribosomes stored in the egg. Compared with other cells, the mouse egg contains onethird to one-fifth as much total RNA (the bulk of which is ribosomal) per unit volume as Xenopus or sea urchin eggs and only about one-sixth as much as a cell from regenerating rat liver (see Piko and Clegg, 1982). The maternal store of ribosomes is reduced by a further 20-25% in the two-cell embryo. Thus the buildup in ribosomes is necessary to provide for the approximately eightfold increase in the overall rate of protein synthesis between the two-cell and blastocyst stages (Brinster et al., 1976; Abreu and Brinster, 1978). An early hypothesis based on circumstantial evidence that the Key Words: Ribosomal protein mRNAs, Blot hybridizamouse egg had a large “hidden” supply of ribosomes in tion, Ribosome biosynthesis, Preimplantation development the form of fibrous lattices could not be substantiated (Piko and Clegg, 1982); instead, the abundant fibrous material that is characteristic for rodent eggs, but is INTRODUCTION quite variable in fine structural appearance according During the growth phase of oogenesis, the mouse to species, most likely represents reserve yolk in these oocyte accumulates a supply of messenger RNA eggs (Szollosi, 1972; Nilsson, 1980). (mRNA), transfer RNA, and ribosomes that supports Ribosome biogenesis depends on the availability of protein synthesis during meiotic maturation and post- a n adequate supply of ribosomal RNA and ribosomal fertilization development through the first cleavage di- protein molecules (see review by Mager, 1988), but how vision (reviewed by Wassarman, 1983; Bachvarova, the synthesis of these components is regulated in mouse 1985; Schultz, 1986). The fully grown, preovulatory oocytes and early embryos is a s yet incompletely underoocyte contains about 0.26 ng of ribosomal RNA stood. Ribosomal RNA synthesis probably ceases, or is (amounting to about 60%of a total RNA content of 0.43 reduced to a low level, toward the end of the major ng), equivalent to about 7 x lo7 ribosomes (Bach- growth phase of the oocyte, when the pronuclei take on varova et al., 1985; Paynton et al., 1988). During mei- a highly compacted, agranular morphology (Chouiotic maturation and embryonic development through the two-cell stage, both ribosomal RNA content and the number of morphologically recognizable ribosomes decline to about 5.5 x lo7 ribosomes in the ovulated egg Received August 23,1991; accepted October 9,1991. and about 4.2 x lo7 ribosomes in the late two-cell em- Address reprint requests to Dr. Lajos Piko, 151-B3, Developmental bryo (Paynton et al., 1988; Piko and Clegg, 1982). The Biology Laboratory, VA Medical Center, Sepulveda, CA 91343. The quantitative changes in the ABSTRACT mRNAs for ribosomal proteins Va, L18a, and S15 were assayed in slot hybridization experiments using labeled cRNA probes with total RNA from late growth-phase oocytes, ovulated eggs, and early embryos through the blastocyst stage. All three mRNAs showed a similar developmental pattern of prevalence, but their copy numbers per oocyte or embryo fluctuated according to developmental stage. There are on an average about 17,000copies of each mRNA in the late growth-phase oocyte; this number drops to one-fifth to one-tenth in the ovulated egg and two-cell embryo but increases rapidly during cleavage to about 25,000 in the eight-cell embryo and about 42,000 in the blastocyst. A comparison of the levels of these mRNAs with the reported rates of ribosomal protein synthesis (LaMarca and Wassarman, 1979) suggests that, in late growth-phase oocytes, ribosomal protein synthesis is regulated primarily at the translational level and is kept low by some factor limiting mRNA utilization. On the other hand, the high rate of ribosome biosynthesis during early embryogenesis from the two-cell stage onward appears to involve the coordinate activation and transcription of ribosomal RNA and ribosomal protein genes coupled with the immediate translational utilization of ribosomal protein mRNAs.

0 1992 WILEY-LISS, INC.

RIBOSOMAL PROTEIN mRNAS IN MOUSE EMBRYOS nard, 1971; Crozet et al., 1981; see Bachvarova, 1985). The earliest stage of embryo development at which ribosomal RNA synthesis can again be detected is the mid- to late two-cell embryo (Knowland and Graham, 1972; Clegg and Piko, 1982). Fully differentiated, fibrillogranular nucleoli (Hillman and Tasca, 1969; Takeuchi and Takeuchi, 1986) and a high level of ribosomal RNA synthesis (Piko, 1970; Clegg and Pik6, 1977) are characteristic for the cleavage stages. A somewhat different pattern has been observed with respect to the synthesis of ribosomal proteins. Despite the probable absence of ribosomal RNA synthesis, ribosomal proteins continue to be synthesized at a low rate, amounting to 1.1-1.5% of total protein synthesis, throughout the period from the late stages of oocyte growth through meiotic maturation; however, the rate of synthesis of ribosomal proteins increases sharply during early embryogenesis as shown by a n approximately ll-fold rise (to about 8%of total protein synthesis) in the eight-cell embryo (LaMarca and Wassarman, 1979). Since oocyte mRNA synthesis ceases upon germinal vesicle breakdown (Wassarman and Letourneau, 1976; Rodman and Bachvarova, 1976) and significant new synthesis of mRNA does not resume until the twocell stage (Clegg and Piko, 1983a), ribosomal protein synthesis must be carried out on maternal mRNA templates a t least through the first cleavage division. However, there is no information at present on the storage of ribosomal protein mRNAs in the egg or the extent to which ribosomal protein synthesis during cleavage is dependent on new mRNAs derived from the embryonic genome. In this communication, we report on the developmental pattern of the prevalence of three ribosomal protein mRNAs during the period from the late stages of oocyte growth through the blastocyst stage. Two of the mRNAs studied encode ribosomal proteins L7a and L18a that form a part of the large (60s) ribosomal subunit, and one mRNA encodes ribosomal protein S15, a constituent of the small (40s) subunit. The cDNA probes used in these experiments were originally obtained in a random cDNA library produced from late two-cell embryos (Taylor and Piko, 1987) and were selected for study because of their apparent low prevalence in the egg but increased abundance during the cleavage stages. They were subsequently identified a s ribosomal protein genes by nucleotide sequencing. The results show that transcripts derived from these genes are in fact quite abundant in nearly fully grown oocytes, but, unlike the bulk of the maternal mRNA, they undergo a n early degradation and are reduced in amount to one-fifth to one-tenth in the ovulated egg. During the two-cell stage, the transcription of the three genes appears to become activated, as indicated by a n approximately tenfold rise in the absolute amounts of their mRNAs between the two-cell and eight-cell stages and a further doubling by the early blastocyst stage. Projected for 70 to 80 ribosomal genes, ribosomal protein mRNAs may account for 8-15% of total mRNA during cleavage. A comparison of the pattern of prevalence of

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ribosomal protein mRNAs with the reported rates of ribosomal protein synthesis suggests that, in late growth-phase oocytes, the rate of ribosomal protein synthesis is controlled primarily at the level of translation and is kept low by some factor limiting mRNA utilization. In the eight-cell embryo, the rise in the level of ribosomal protein synthesis parallels the accumulation of ribosomal protein mRNAs and that of ribosomal RNA. The data support a model according to which the high rate of ribosome biosynthesis in the mouse zygote from the two-cell stage onward involves the coordinate activation and transcription of ribosomal RNA and ribosomal protein genes as well a s the immediate translational utilization of ribosomal protein mRNAs.

MATERIALS AND METHODS cDNA Clones All three cDNA clones were obtained in a random cDNA library prepared in pUC8 plasmid vector from late two-cell mouse embryos and were originally designated A19, B16, and D16 (Taylor and Piko, 1987). Clone A19 contains a cDNA insert of about 0.3 kb; clone B16, about 0.5 kb; and clone D16, about 0.8 kb. The cDNA inserts were fully or partially sequenced using either the chemical degradation method of Maxam and Gilbert (1980) or the Sequenase reaction ( U S . Biochemical Corp.; Zhang et al., 1988). Sequence comparisons were made by searching the GenBank gene library (release 64) using version 5.0 of the Hitachi HIBIO DNASIS program. This search identified the three cDNA clones as the mouse homologues for ribosomal protein genes L18a (clone A19), L7a (clone D16), and ,515 (clone B16).

Oocytes and Embryos Large oocytes (65-70 pm in diameter without the zona pellucida) were isolated from the ovaries of 15day-old CD2Fl mice (BALBk female x DBM2 male; Charles River Breeding Laboratories) and freed of follicle cells as described (Pik6 and Taylor, 1987). Early embryos up to the blastocyst stage were recovered directly from superovulated and mated CD2Fl mice. Late blastocysts were obtained by culturing early blastocysts in Brinster’s pyruvate-lactate medium for 24 hr. Batches of oocytes and embryos were lysed in 300 p1 of 0.5% sodium dodecyl sulfate (SDS) and 500 pg per ml proteinase K (Bethesda Research Laboratories) in 0.1 M NaC1,50 mM Tris HC1, pH 7.5,5 mM EDTA. About 90,000 cpm of 3H-labeled A cRNA was added to each lysate as a recovery marker.

RNA Extraction Total RNA from embryo lysates was isolated by phenol-chloroform extraction, Nensorb-20 (Dupont-NEN) column chromatography and digestion with RNase-free DNase as described elsewhere (Taylor and Piko, 1987). A poly(A)+ RNA-rich fraction from F9 mouse embryo-

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K.D. TAYLOR AND L. PIKO

nal carcinoma cells was obtained in a previous work (Pikb and Taylor, 1987).

Southern Blots BALB/c mouse liver DNA was isolated by digestion of a nuclear fraction with SDS/proteinase K and buoyant density centrifugation in a CsCl gradient. Samples of 5 pg nuclear DNA were digested with the restriction endonuclease EcoRI or HindIII and, after electrophoresis on a 0.7% agarose gel, blotted onto nitrocellulose filters (Maniatis et al., 1982). The blots were hybridized with (3,000 Ci/mmol; cDNA probes labeled with [cx-~’P]~TTP ICN) using a random priming procedure (BRL).

3’ end of the published sequence of the mRNA for the rat ribosomal protein L18a (Aoyama et al., 19891, thus identifying the A19 clone as the mouse homologue of the L18a gene. Fipure 1 shows the 3’ untranslated region (UTR) in t h e 1 1 9 clone together with the reported (truncated) 3’ UTR of the ra t L18a mRNA. From the rat L18a cDNA sequenced by Aoyama et al. (1989) and the length of the 3‘ UTR in A19, the full length of the L18a mRNA can be estimated to be about 590 nucleotides without the poly(A) tail. In clone D16, three noncontiguous segments of the cDNA insert totaling 0.52 kb were sequenced, including a 3‘ terminal segment of the cDNA. The nucleotide sequence of these regions (not shown) shared a 96% homology with the corresponding regions of the cDNA for rat ribosomal protein L7a (Nakamura et al., 1989) and was virtually identical to the transcript of the mouse Surf-3 gene (Huxley et al., 1988)that was subsequently shown to encode the mouse L7a ribosomal protein (Giallongo et al., 1989). These data identify clone D19 as a derivative of the mRNA for ribosomal protein L7a. The full length of the mouse L7a gene transcript, excluding the poly(A) tail, is 866 or 870 bases, with a 5‘ UTR of 22 or 26 bases, a coding sequence of 798 bases, and a 3’ UTR of 46 bases (Huxley et al., 1988; Huxley and Fried, 1990). Since, in clone D16, the cDNA insert ends with the termination codon at the 3’ end of the coding region and lacks a 3’ UTR, the 0.8 kb insert in this clone probably contains most or all of the coding sequence of the L7a mRNA. The cDNA insert in clone B16 has been fully sequenced and found to be identical to the mouse rig (for “rat insulinoma gene”) gene (Sugawara et al., 1990; Taylor and Piko, 1991). The rig gene has recently been identified as the gene encoding ribosomal protein S15 (Kitagawa et al., 1991). Although the developmental pattern of expression of the rig gene in early mouse embryos has been reported previously (Taylor and Piko, 19911, some of the data are included here (see Table 1) to provide a more comprehensive picture of ribosomal protein gene expression during early mouse embryogenesis. In the remainder of this paper, the three cDNA clones will be referred to by the name of the corresponding ribosomal protein: L18a (for A19), L7a (D16) and ,315 (B16lrZg).

Blot Hybridization of RNA Slot blot and Northern blot hybridizations were carried out a s described previously (Taylor and Pikb,1990, 1991). Sense RNA standards and labeled RNA probes were synthesized on cDNA subcloned in the plasmid pGEM-4Z (Promega). Probe RNAs were labeled with [cx-~’P]UTP(1,000 Ci/mmol; ICN) to a specific activity of about 1.6 x lo6 d p d n g . The amounts of standard RNAs were calculated from the incorporation of [3H]ATP(1.0 Ci/mmol; Amersham). RNA samples were denatured in 25 mM Na phosphate, pH 7 , 5 mM EDTA, 2.2 M formaldehyde, 50% formamide at 65°C for 5 min and either dotted directly (in 2 0 SSC) ~ onto GeneScreen Plus filters (Dupont-NEN) or transferred to these filters after electrophoresis on agarosel formaldehyde slab gels (Maniatis et al., 1982). The filters were ultraviolet (UV) cross linked as described (Taylor and Pikb, 1990) and hybridized with 10 ng/ml probe RNA for 18 h r a t 65°C in 50% formamide, 1 M NaC1, 50 mM Na phosphate, pH 7, 10 mM EDTA, 1% SDS, 5X Denhardt’s solution, 200 pg per ml herring sperm DNA, 20 pg per ml poly(A), 50 k g per ml yeast tRNA. After a brief rinse, the filters were treated with 130 pg/ml RNase A and 60 U/ml RNase T1 (Boehringer-Mannheim) in 0.375 M NaC1, 75 mM Tris HCl, pH 7.5, 5 mM EDTA for 5 min a t room temperature, washed for 2 hr at 68°C in several changes of 25 mM Na phosphate, pH 7 , 4 mM EDTA, 1%SDS, and autoradiographed at -70°C on preflashed X-ray films (Dupont Chronex No. 4) with intensifying screens. The density of the slots was determined by scanning the autoradiographs with a Biorad model 1650 densitometer. The Southern and Northern Blots number of RNA molecules in the samples was calcuHybridization of Southern blots of EcoRI-digested lated by comparison to the standard RNA slots as denuclear DNA from mouse liver with the L18a cDNA scribed (Taylor and Piko, 1990). probe yielded about six major and several minor bands (Fig. 2A, lane l ) , suggesting that the L18a gene is RESULTS present in multiple copies. The same experiment using Identification of cDNA Clones the L7a cDNA probe revealed 20 to 30 bands (Fig. 2A, After the cDNA clones were partially or fully se- lane 2). A similar number of bands was obtained for quenced, their identity was determined by searching each gene with mouse nuclear DNA digested with for homologous sequences in the GenBank gene library. HindIII (not shown). Gel blot hybridization of a In clone A19, a 0.25 kb segment at the 3‘ end of the poly(A)+ RNA fraction from F9 mouse embryonal carcicDNA insert was sequenced, including a poly(A) tail of noma cells revealed a discrete, abundant RNA species (A)15.The nucleotide sequence of this segment preced- with each probe; the molecular weight of the RNA was ing the poly(A) tail showed about 97% homology to the about 0.75 kb with the L18a probe (Fig. 2B, lane 1)and

RIBOSOMAL PROTEIN mRNAS IN MOUSE EMBRYOS Mouse A19

-TAGACA-CAGAGACCCACTGAATAAAAACTTGAGACTGTC(A)15

Rat L18a

-TAGACACCAGAGACCCACTGAATAAAAG

185

........................... . . . . . . . . .................

Fig. 1. Nucleotide sequence of the 3’ untranslated region (3’ UTR) of the cDNA insert in clone A19 compared with the published sequence of the 3’ UTR (truncated)of a cDNA for rat ribosomal protein L18a (Aoyama et al., 1989).Colons indicate nucleotide homology; a dash was introduced in the A19 sequence to obtain optimum alignment.

TABLE 1. QuantitativeChanges in the mRNAs for Ribosomal Proteins L7a, L18a, and 515 in Mouse Oocytes and Early Embryos Hours after HCG injectiona

Stage of No. of mRNA per embryob development L7a L18a S15 Large oocyte 1.5 f 0.1 X lo4 2.0 f 0.4 X lo4 1.6 f 0.2 X lo4 21 Unfertilized egg 3.4 f 0.5 X lo3 2.8 f 0.3 X lo3 2.1 k 0.1 X lo3 46 Late two-cell 1.7 f 0.1 x 103 2.5 k 0.4 x 103 2.1 0.4 x 103 70 Eight-cell 3.4 t 0.1x 104 1.8 f 0.2 x 104 2.5 f 0.3 x 104 94 Early blastocyst 5.2 f 1.1 X lo4 3.6 f 0.4 X lo4 3.7 f 0.2 X lo4 118 Late blastocvst 5.2 & 0.5 X lo4 4.1 f 0.2 X lo4 4.1 f 0.6 X lo4 aApproximate age of embryos. Ovulation occurs at 12-14 hr, first cleavage at 30-32 hr, and second cleavage a t 51-53 hr after the injection of human chorionic gonadotropin (HCG).Large (nearly fully grown) oocytes were recovered from the ovaries of 15-day-old mice. bThenumber of mRNA molecules per embryo was calculated from densitometric tracings of autoradiographs obtained in slot hybridization experiments of total embryo RNA in relation to RNA standards synthesized for each mRNA (Fig. 3). The data represent the average of two (L7a)or three (L18a and S15) determinations fSEM. The data for the S15 ribosomal protein mRNA, formerly termed rig mRNA, are from Taylor and Pikd (1991).

+

about 1.0 kb with the L7a probe (Fig. 2B, lane 2), which is in agreement with the estimated lengths of these mRNAs (see above) plus poly(A) tails. A single RNA species of the same size was obtained with each probe also in Northern blots of total RNA from about 20 mouse blastocysts (not shown).

tle if any additional increase in late blastocysts, presumably because embryo development is arrested a t this stage in the pyruvate-lactate culture medium used.

DISCUSSION

The mammalian ribosome contains stoichiometric amounts of 70-80 distinct proteins and four RNA speDevelopmental Pattern of Prevalence of cies: 18s RNA in the 40s ribosomal subunit and 28S, Ribosomal Protein mRNAs 5S, and 5.8s RNAs in the 6 0 s subunit (Wool, 1986). In The steady-state amounts of the L18a and L7a the mammalian genome, the genes for ribosomal promRNAs a t different stages of development were as- teins are present as dispersed multigene families, with sayed in slot hybridization experiments (Fig. 3). The from about seven to more than 20 members per gene filters were treated with RNase A and T1 after hybrid- family (Monk et al., 1981; D’Eustachio e t al., 1981). ization, in order to facilitate quantitation and to reduce Typically, there is only one intron-containing gene that possible nonspecific binding of the probes. The results is transcriptionally active for each ribosomal protein, are summarized in Table 1, which also includes data for whereas the remainder are inactive, processed the S15 (formerly termed rig)mRNA that was assayed pseudogenes (Dudov and Perry, 1984; Davies et al., previously (Taylor and Piko, 1991). The three riboso- 1989). The identity of the cDNA clones used in this study mal protein mRNAs show a similar developmental pattern of expression. They are relatively abundant has been established on the basis of their nucleotide (15,000-20,000 copies) in nearly fully grown oocytes homology to published sequences of ribosomal protein but are reduced in amount to one-fifth to one-tenth genes. Genome blots for ribosomal protein genes L7a (2,000 to 3,000 copies) in the unfertilized egg. Although and L18a in the present work (Fig. 2A) and for the S15 the copy numbers of the three mRNAs in the two-cell gene in a previous work (Taylor and Pik6,1991) are in embryo are similar to t h a t in the egg, their relative agreement with multiple copies for these genes in the prevalence is increased about threefold because of the mouse genome as reported previously (Huxley e t al., large-scale degradation of maternal poly(A)+ mRNA 1988; Aoyama e t al., 1989; Shiga e t al., 1990).Northern during the two-cell stage (Clegg and Piko, 198313). blot experiments have confirmed the presence of a sinThere is a steep rise in mRNA copy number in eight- gle RNA transcript of the appropriate molecular weight cell embryos (to a n average of about 25,000 copies) and (as derived from the sequence of the corresponding early blastocysts (about 42,000 copies), but there is lit- cDNAs) in F9 mouse embryonal carcinoma cells and

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kb 12.29.2 7.1 5.1 4.1 3.1 -

K.D. TAYLOR AND L. PIKO

’

A kb

I

B

7.54.4-

2.4-

2.0-

1.4-

1 .o-

0.55-

B

A

2

~ 1 . kb 0 ~ 0 . 7 hb 5

Fig. 2. A. Southern blot analysis of the mouse ribosomal protein genes L18a (lane 1) and L7a (lane 2). Samples of 5 kg mouse liver nuclear DNA were digested with EcoRI, blotted, and hybridized with 10 ngiml probe DNA labeled with 32Pto about 5 x lo6 dpm/ng using a random priming procedure. Autoradiography was for 24 hr. The migration of standard DNA bands (DNA ladder, BRL) is shown at left. B. Gel blot hybridization of a poly(A)’ RNA fraction (60 ng/lane) from F9 mouse embryonal carcinoma cells with 32P-labeled cRNA probes (about 1.6 x lo6 dpm/ng; 10 ng probe/ml) to cDNA clones L18a (lane 1) and L7a (lane 2). The blot in lane 1 was treated with RNase A and T1 after hybridization; the blot in lane 2 was not. Autoradiography was for 24 hr. The migration of the RNA size standards (RNA ladder, BRL, including also a 0.55 kb cRNA marker) is shown a t left.

mouse blastocysts for each of the three genes studied (Fig. 2B; Taylor and Piko, 1991). The results of quantitative dot hybridization experiments (Fig. 3, Table 1)indicate that 1)the developmental pattern of prevalence is similar for the three ribosomal protein mRNAs studied and 2) the absolute levels of these mRNAs and their relative abundance in relation to the total mRNA population vary markedly depending on developmental stage. The levels of all three mRNAs are relatively high in nearly fully grown oocytes but are reduced to one-fifth to one-tenth in the ovulated egg, whereas, during this period, there is a loss of only about 30% in the mRNA population a s a whole (Paynton et al., 1988).By the late two-cell stage, total mRNA content is reduced by a further 70% (Piko and Clegg, 1982), but there is little change in the absolute amounts of the ribosomal protein mRNAs. As a result of this differential degradation, the relative abundance of ribosomal protein mRNAs is greatly reduced in the egg (to about 0.01% of the total poly(A)+ RNA population a t this stage; Clegg and Piko, 1983b), but i t is enhanced severalfold in the two-cell embryo. To what extent the level of ribosomal protein mRNAs a t the two-cell stage is attributable to maternal storage vs. new synthesis by the embryo is not known. However, the sharp increase in the amounts of ribosomal protein mRNAs during cleavage clearly results from new transcription by the embryonic genome. In the eight-cell and blastocyst stages, the relative abundance of the individual ribosomal mRNAs in relation to total poly(A)+ RNA rises to 0.1-0.2%; assuming a similar average abundance for the 70-80 ribosomal protein

fg

No. f g

N 0.

320

17 3 2 0

10 LB

160

20 160

10 EB

80

21

80

I0 8 C

40

33

40

23 2 C

20

17

20

20 1c

I0

17

I0

14 LO

Fig. 3. Slot hybridization assay of ribosomal protein mRNAs L18a (filterA) and L7a (filterB) in mouse oocytes and embryos a t different stages of development. Total embryo RNA (DNase-treated)was dotted onto Genescreen Plus filters and hybridized with 32P-labeledcRNA probes (10 ngiml; about 1.6 x lo6 d p d n g ) ; a 1:l dilution series of the corresponding sense RNA standard was also dotted onto each filter. The amount of standard RNA dotted is shown a t the left side of the filter, and the number of embryos used is shown at right. The filters were treated with RNase A and T1 after hybridization and exposed for autoradiography for 4 days. Stages of embryo development: LO, large oocytes, 65-70 km in diameter, obtained from 15-day-old mice; l C , unfertilized eggs; 2C, two-cell embryos; 8C, eight-cell embryos; EB, early blastocysts (32 cells); and LB, late blastocysts (64 cells). For the chronological age of the embryos and a summary of the results, see Table 1.

mRNAs, the combined contribution of the latter would amount to 8-15% of the mRNA population as a whole during cleavage. To gain some insight into the utilization of ribosomal protein mRNAs in mouse oocytes and early embryos, it is interesting to compare the levels of the three mRNAs measured in this study with the reported rates of synthesis for a total of twelve ribosomal proteins (LaMarca and Wassarman, 1979). The average rates of synthesis per hour were about 1.1 x lo5 and 1.7 x lo5 protein molecules in late growth-phase and preovulatory oocytes, respectively, and about lo5 molecules in unfertilized eggs; projected for 70 ribosomal proteins, these rates account for about 1.5% of the total protein synthesis in oocytes and 1.1% in eggs. Surprisingly, the rates of synthesis of individual ribosomal proteins varied as much a s tenfold at all stages examined, suggesting that they are not under tight coordinate control, although newly synthesized ribosomal proteins accumulated in the oocyte’s germinal vesicle in near-equimolar amounts (LaMarca and Wassarman, 1979, 1984; Wassarman, 1983). The pattern of prevalence of ribosomal protein mRNAs obtained in the present study, showing a steep decline between the late growth-stage oocyte and the ovulated egg but little variation among individual mRNAs, suggests t h a t the observed rates of synthesis of ribosomal proteins during this period are not dependent on mRNA concentration but are controlled primarily at the level of translation. A comparison of the

RIBOSOMAL PROTEIN mRNAS IN MOUSE EMBRYOS protein synthetic rates in late growth-phase oocytes (LaMarca and Wassarman, 1979) with the corresponding levels of the three ribosomal protein mRNAs (about 17,000 copies) indicates that the efficiency of utilization of ribosomal protein mRNAs is very low at this stage, yielding a n average of only about 0.11 (0.3-0.26) protein molecules min-l/mRNA. This rate of translation is about one-tenth that calculated in fully grown oocytes for actin mRNA (Taylor and Piko, 1990) or total polysomal mRNA (Davidson, 1986, pp 401413). A possible mechanism of translational control may be that some factorb) in the protein synthesis initiation cycle (Rhoads, 1988) becomes rate limiting toward the end of oocyte growth, reducing the rate of ribosomal protein synthesis despite the abundance of ribosomal protein mRNAs at this stage. The limiting factor may affect individual mRNAs to a different extent, leading to a variation in the rate of translation. It is now well established that translational control plays a major role in regulating the expression of ribosomal protein genes in eukaryotic cells to satisfy the physiological need of cells for ribosome biosynthesis, for example, in early Xenopus embryos (Amaldi et al., 1989) and glucocorticoidrepressed mouse lymphosarcoma cells (Meyuhas et al., 1990; see Mager, 1988). Recent evidence strongly suggests that the translational regulation is dependent on the 5’ terminal oligopyrimidine tract, which is a common feature of ribosomal protein mRNAs in vertebrate cells, but it is not clear whether the control mechanism involves a specific trans-acting factor or some component of the general protein-synthetic apparatus (Levy et al., 1991). A different type of regulation, where the transcription of ribosomal protein mRNAs is closely coupled with their translation, appears to be prevalent during the cleavage stages of mouse embryo development. In the mid- to late two-cell embryo, the transcription and processing of ribosomal RNA genes become activated (Clegg and Piko, 1982) and this seems to be accompanied by the coordinate expression of other ribosomeassociated genes such as the genes for U3 small nuclear RNAs that are thought to be involved in ribosomal RNA processing (Prather et al., 1990) and the ribosomal protein genes (present study). The appearance of a fibrillogranular component in the nucleoli of late twocell embryos (Hillman and Tasca, 1969) corroborates the view that ribosome biosynthesis begins at this stage. Between the two-cell and eight-cell stages, the levels of ribosomal protein mRNAs increase about 12fold (Table 1).A similar rise, to about 1.3 x lo6 molecules hr-’/embryo, is observed in the absolute rates of synthesis of ribosomal proteins that are now synthesized in more nearly equimolar amounts (LaMarca and Wassarman, 1979). The estimated molar accumulation of ribosomal proteins is comparable, within a factor of two, to that of ribosomal RNA and is also commensurate with the observed increase in the number of ribosomes a t this stage (Piko and Clegg, 1982).These observations suggest that the newly transcribed ribosomal protein mRNAs in early mouse embryos are immedi-

187

ately utilized for translation and that the rate of synthesis of ribosomal proteins is coordinated with that of ribosomal RNA. A different pattern of regulation, involving a delayed utilization of ribosomal protein mRNAs, is observed in early Xenopus embryos: in this species, new synthesis and accumulation of ribosomal protein mRNAs begin at the blastula stage, but the mRNAs are not recruited into polysomes until the tail bud stage more than 20 h r later, when the production of new ribosomes is required (Amaldi et al., 1989; Mariottini and Amaldi, 1990). However, it should be noted that, on the basis of a rate of ribosomal protein synthesis of about 1.3 x lo6 molecules hr-’/embryo (see above) and a n estimated average mRNA content of about 25,000 (Table l), the utilization of ribosomal protein mRNAs in the eight-cell mouse embryo is still relatively inefficient, yielding about one protein molecule min-’/mRNA compared with a n estimated translational efficiency at this stage for the mRNA population as a whole of about four polypeptides min-’/mRNA (Taylor and Piko, 1990). This apparent low efficiency of translation is in line with the observation that typically only a fraction of ribosomal protein mRNAs is associated with polysomes even in normal exponentially growing cells (Mayuhas et al., 1990). The efficiency of translation of ribosomal protein mRNAs in the mouse embryo is likely to be higher at the blastocyst stage, when ribosome production per embryo is estimated to rise about fourfold compared with the eight-cell stage (Piko and Clegg, 1982), whereas the average content of the three ribosomal protein mRNAs studied has risen only about twofold (Table 1).

ACKNOWLEDGMENTS We are grateful to Linda Western for sequencing the cDNA clones and Miriam Pillos for technical assistance. This work was supported by Public Health Service research grant HD-19691 from the National Institute of Child Health and Human Development. REFERENCES Abreu SL, Brinster RL (1978): Synthesis of tubulin and actin during the preimplantation development of the mouse. Exp Cell Res 114:135-141. Amaldi F, Bozzoni I, Beccari E, Pierandrei-Amaldi P (1989): Expression of ribosomal protein genes and regulation of ribosome biosynthesis in Xenopus development. Trends Biochem Sci 14:175-178. Aoyama Y, Chan Y-L, Meyuhas 0,Wool IG (1989):The primary structure of rat ribosomal protein L18a. FEBS Lett 247:242-246. Bachvarova R (1985): Gene expression during oogenesis and oocyte development in mammals. In LW Browder (ed): “Developmental Biology. A Comprehensive Synthesis.” New York: Plenum, pp 453524. Bachvarova R, De Leon V, Johnson A, Kaplan G, Paynton BV (1985): Changes in total RNA, polyadenylated RNA, and actin mRNA during meiotic maturation of mouse oocytes. Dev Biol 108:325-331. Brinster RL, Wiebold JL, Brunner S (1976): Protein metabolism in preimplanted mouse ova. Dev Biol51:215-224. Chouinard LA (1971): A light- and electron-microscope study of the nucleolus during growth of the oocyte in the prepubertal mouse. J Cell Sci 9:637463. Clegg KB, Piko L (1977): Size and specific activity of the UTP pool and overall rates of RNA synthesis in early mouse embryos. Dev Biol 58:7&95.

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