IlEVEI,OPMENT.4I,

I~IOLOGY

138, 247-855

(1990)

U3 snRNPs and Nucleolar Development during Oocyte Maturation, Fertilization and Early Embryogenesis in the Mouse: U3 snRNA and snRNPs Are Not Regulated Coordinate with Other snRNAs and snRNPs R. PRATHEH,*,' C. STMEKI.Y,* W. P. MARZLUFF,?

G.

SCFIATTEN,*”

W. L.

DEAN,+

D. AND

R. PILCH,t G. A.

S. M.

LOBO,~

SCFIULTZ~

U3 small nuclear riboqucleic acids (snRNA) and U3 small nuclear riborlucleoprotrin isnRNP), which are thought to be responsible for ribosomal RNA processing, are quantitated and localized during oocyte maturation, fertilization, and early embryogenesis in the mouse. On the basis of Northern blot and nuclease protection experiments, it is estimated that there are about 5 x 10’ U3 snRNA molecules in an ovulated oocytc and in a two-cell embryo. This number then incrcascs roughly 50-fold to 2.7 X 10” molecules per embryo by the blastocyst stage. At all stages of development U3 snRNP antigens colocalize with nucleoli, as defined by differential interference contrast microscopy and an antibody to a nucleolar epitope. The synthesis and distribution of 1J3 snRNA and rJ3 snRNP follow a pattern independent. from other major U snRNPs and snRNAs. I I’Wil Ar:~,lrmlr l’fY.+d. Im INTRODUCTION

Ribonucleic acid (RNA) synthesis increases two- to threefold as mouse oocytes progress from primordial oocytes to fully grown oocytes (Moore et nl., 1974; Fourtroy, 1982). Ribosomal RNA production (18 S and 28 S) during the growth phase of oogenesis has been measured at 0.01 pg/min (Bachvarova, 1981). When the oocyte acquires multiple layers of follicular cells, the rate of rRNA synthesis declines (Kaplan et ul., 1982). The amount of RNA accumulated in the fully grown oocyte is 0.45 to 0.6 ng (Sternlicht and Schultz, 1981; Kaplan pt al., 1982) of which 65% to 70% is stable rRNA. Once a fully grown oocyte is hormonally stimulated, RNA synthesis ceases and the germinal vesicle breaks down (GVBD). Meiotic maturation then arrests at metaphase II. The arrest is terminated when the oocytc is activated, then the second polar body is emitted, and pronuclei develop. At first mitosis, about 20 hr post,-sperm penetration, the male and female genomes mix and after division segregate to the two sibling blastomeres. The two-cell stage is characterized by the reactivation of transcription and the production of all classes of RNA although rRNA synthesis is not detectable until ’ Present address: Department of Animal Science, LJniversity of Missouri, Columbia, MO 65211. ‘To whom correspondence should be addressed at Integrated Microscopy Resource, 1117 W. Johnson St., Iinivcrsity of Wisconsin, Madison I WI c,53706.

the late two-cell stage (Piko and Clegg, 1982; Clegg and Piko, 1983). The amount of rRNA production and accumulation of new ribosomes then continually increases from the late two-cell stage to the blastocyst (Hillman and Tasca, 1969; Piko and Clegg, 1982). The processing of the primary RNA transcripts is facilitated by small nuclear ribonucleic acids (snRNAs) and associated small ribonucleoproteins (snRNP) (Maniatis and Reed, 1987). The more abundant snRNAs have been characterized and named UI, U2, U3, U4, U5, and U6. Ul, U2, U4, U5, and U6 are involved in premRNA splicing (Maniatis and Reed, 1987), while U3 snRNA and associated proteins are thought to be involved in a processing event near the 3’ end of 28 S pre-rRNA (Parker and Steitz, 198’7). Ul, U2, U4, U5, and U6 remain relatively constant per embryo from ovulation to the two-cell stage, but increase 3- to lo-fold by the blastocyst stage (Lobo Pf al., 1988; Dean et al., 1989). Here we show that although the number of U3 snRNA molecules in mouse oocytes and embryos are lower than the corresponding IT snRNAs (Ul, IJ2, U4, U5, IJ6) for pre-mRNA splicing, U3 snRNPs are detectable in all interphase nuclei during early embryogenesis. The number of U3 snRNA molecules per embryo increases markedly between the two-cell and blastocyst stage, when rRNA is actively synthesized. These changes in U3 snRNA abundance are correlated with the changes in nucleolar structure and function that occur during early embryogenesis in the mouse.

DEVELWMENTALBIOLOGY

248 MATERIALS

AND

METHODS

Superovulation, mating, and recovery of ICR Swiss albino oocytes and embryos were accomplished as previously described (Lobo et al., 1988). Measurement of U3 snRNA. Total RNA was extracted from mouse oocytes and embryos as described previously (Lobo et al., 1988; Dean et al., 1989). Probes for U3 snRNA and Ul snRNA (for comparison) were hybridized in parallel to aliquots of the same RNA samples in both Northern blot and nuclease protection experiments. For Ul snRNA assays, antisense transcripts of a 394-bp EcoRVSstII fragment of mouse genomic DNA (that contains a complete 165 bp mouse Ulb-2 gene) inserted into an Sp65 plasmid were generated following linearization with Hind111 exactly as described by Lobo et al. (1988). The probe used for U3 snRNA measurements was a 340-bp rat genomic DNA fragment (EcoRI/HindIII) encoding a 214-nucleotide U3 gene that was inserted into Sp64 and Sp65 transcription vectors (Stroke and Weiner, 1985). Antisense transcripts were produced upon linearization of Sp65 with Hind111 and transcription with Sp6 polymerase in the presence of either [a-““P]UTP (800 Ci/mmole, NEN DuPont) or [ol-““S]UTP (1300 Ci/mmole, NEN-DuPont). All other procedures for generating radiolabeled probes, Northern blots, hybridization, and nuclease protection assays were carried out as described by Lobo ef ab. (1988). Autoradiograms were scanned with a densitometer to quantitate the relative amounts of Ul and U3 snRNAs. To measure U3 RNA abundance in absolute terms, the U3 RNA content of a nuclear RNA preparation from SCCl embryonal carcinoma (EC) cells was established by hybridization of radiolabeled antisense transcripts to a slot blot of increasing amounts of EC cell RNA (based on AzGameasurements) in parallel with known amounts of sense-strand U3 RNA (based on AzCiomeasurements) produced in vifro from the transcription vector. In this experiment, 10 ng of EC cell RNA gave the same hybridization signal as 6.6 pg of sense-strand U3 RNA. Similar approaches have been used to measure the Ul, U2, and U6 snRNA content of the EC cell nuclear preparation (Lobo et al., 1988; Dean et al, 1989). The EC cell preparation was then included as a standard on Northern blots to compare the intensity of hybridization of antisense probes to RNA from mouse oocytes and early embryos with known amounts of “U” RNA. The numbers of Ul and U3 RNA molecules were calculated following correction of efficiency of extraction (69 + 2%) taking into account the sizes of 165 and 214 nucleotides, respectively. Antibodies and immuno~uoresce~~ce microscopy. Human antisera JH (D16), which recognizes a 34- to 36-kDa protein and precipitates U3 RNPs and is thereEmbryos

ad

culture.

V0~~~~~138.1990

fore thought to recognize fibrillarin (Parker and Steitz, 1987) (a gift from Drs. K. Parker and J. Steitz), was diluted 1:lOOO in PBS for immunolocalization. Human anti-Sm serum that recognizes Ul, U2, U4, U5, and U6 snRNPs was diluted 1:50 with PBS for immunolocalization. Another human antiserum that also recognizes nucleolar proteins (34 kDa, ~18.5, and precipitates U3 RNPs) (Lischwe et al., 1985; Ochs et ah, 1985) in human fibroblast tissue culture cells (a gift from Dr. G. G. Maul, The Wistar Institute) (Maul and Yasuda, 1989) was diluted 1:50 in PBS for immunolocalization. DNA was imaged with the Hoechst dye 33258 added to the penultimate PBS rinse. Mouse oocytes and embryos were processed and permeabilized for indirect immunofluorescence microscopy as described previously (Schatten et al., 1985) except that the cells were fixed in 10 mM dimethyl-3,3’-dithiobispropionimidate dihydrochloride (Pierce Chemical, IL) for U3 localization with antibody JH. Time-lapse video microscopy. Pronuclear development and nucleolar formation was recorded live in time-lapse following the methods of Schatten and Htilser (1983). Oocytes were collected, inseminated, and cultured for 3 hr and zonae pellucidae were removed with 0.5% pronase. A group of 20 denuded eggs was placed in a Dvorak-Stotler Controlled Environment Culture System (Nicholson Precision Instruments, MD) and cultured under 5% CO, at 37°C on the stage of a Zeiss IM microscope equipped with differential interference contrast optics. Images were captured using a Panasonic video camera and recorded on 4 in videotape at 30-set intervals. A time-date generator was placed inline to ensure correct time postinsemination. Frames selected for publication were photographed directly from the video monitor. RESULTS

Localization of snRNP and nucleolar antigens in fizouse embryogenesis. U3 snRNP is immunocytochemitally localized exclusively in the nucleoli of immature oocytes at the germinal vesicle stage (Figs. lB, U3 snRNP, and 1A for corresponding DNA fluorescence image). At germinal vesicle breakdown, the nucleolar staining disappears and the U3 antigen is no longer localized near the chromatin/chromosomes and appears to have dispersed throughout the cytoplasm since the background fluorescence is brighter (Fig. 1D). After fertilization, most of the U3 is localized in the forming nucleoli within the male and female pronuclei but there is also some diffuse cytoplasmic fluorescence (Fig. 1G). In early pronucleate stage zygotes, numerous nucleolar swellings are delineated with the U3 antibody (Fig. 1F). Toward the end of the first interphase U3 is

snRNP localization in mouse oocytes, zygotes, and embryos. Anti-U3 snRNP antibody staining (B, D, G, I, K ) and correFIG. 1. Anti-U3 DNA localization (A, C, F, H, J). (A, B) An intact germinal vesicle in a preovulatory oocgte. IJ3 snRNP is localized in the nlucleolus. (C, sponding D) IJnfer tilized oocyte arrested at second metaphase of meiosis. U3 is not detected in association with the chromosomes thl augh diffuse cgtoplasr nit fluorescence is noted. (E, F, G) Early pronuclear stage, 18 hr post-hCG. After insemination the U3 antibody deter :ts nucleolar particles within the enlarging male and female pronuclei. At the end of first interphase the U3 becomes patchy and punctate as it disperses stage, 41 hr post-hCG. Nucleoli strongly bind the U3 antibody. (J, Kj Morula stage, 88 hr through0 lut the nucleus. (E) DIC. (H. I) Mid two-cell post-hCC 1 shows U3 distribution restricted exclusively to the nucleolus of each interphase blastomere nucleus. Bars = 10 pm.

250

PRATHEH

ET AL.

US snRNA

and

distinguished as patches of staining surrounding welldeveloped nucleolar structures. U3 becomes undetectable at first mitosis. At interphase of the two-cell stage (Fig. 11) and in morulae (Fig, lK), U3 is again restricted to nucleoli in each blastomere nucleus. Cytoplasmic U3 fluorescence is noted at the morula stage. For a comparison of the behavior of U3 versus other snRNPs and to trace the differing behaviors of these snRNPs during the cell cycle, two other antibodies are evaluated: anti-Sm which recognizes major non-U3 snRNP antigens and an antibody specific for the nucleolar antigen fibrillarin. The non-U3 snRNPs are localized in the germinal vesicle of immature oocytes (Fig. 2B) and aggregate into tight foci during germinal vesicle breakdown (Fig. 2D). Non-U3 snRNPs are not detected in arrested unfertilized oocytes (Fig. 2F) and are localized after sperm incorporation, in both the developing pronuclei. Initially they appear concentrated at the outer faces of the pronuclei (Fig. 2H) and later are found throughout the nucleoplasm (data not shown). After first division, snRNPs are found throughout the nuclei, and they appear to be excluded from the nucleolar regions (Fig. 2J). At third mitosis, (Fig. 2K) the non-U3 antigens are undetectable at anaphase (data not shown) and reaccumulate in the nuclear region at telophase (Fig. 2L). Later preimplantation stages follow a similar pattern (data not shown). Time-lapse video microscopy pronuclear

of nucleolar formation in stage eggs. At 3 hr postinsemination, ferti-

lized oocytes could be discriminated from unfertilized ones by the presence of a sperm tail bound to the vitelline membrane and a second polar body (Fig. 3A). An hour later, small nucleolar granules appear (arrows, Fig. 3B) and are clustered at the site of the decondensing male and female chromatins. During the next 30 min, as the male and female pronuclei enlarge, a number of these small granules fuse to form a single predominate nucleolus within each pronucleus (Figs. 3C to 3F, arrows). Changes in the amount of U3 snRNA during early mouse development. The amounts of U3 snRNA and Ul

snRNA

are compared

in parallel

FIG. 2. Anti-Sm snRNP (Ul, U2, localization (A, C, E, G, I, K) and fluorescence appears patchy in the B). At GVBD the snRNPs aggregate oocytes Sm is not detected, perhaps Sm antibody (G, H) and it disperses Sm antigen is distributed throughout early four-cell stage embryo (66 hr chromosomes. This cycle of antigen

Northern

blot experi-

snRN~?s

during

Mouse

DPvehpment

251

ments in Fig. 4A. The amount of U3 snRNA present in samples of roughly 200 oocytes and two-cell embryos was insufficient to yield a detectable hybridization signal when the blot exposure was such that the signal for U3 snRNA in the eight-cell sample was visible and the blastocyst signal for U3 snRNA was comparable to Ul snRNA (Fig. 4A). Hybridization signals for U3 snRNA were obtained, however, when RNA from 1000 or more oocytes or two-cell embryos was analyzed. The intensity of hybridization from 1000 oocytes or two-cell embryos was about one-half as strong as the signal from 50 blastocysts (lanes E-2, 2-2, and B-2 in Fig. 4A). From densitometric measurements the amount of U3 snRNA in the unfertilized oocyte was estimated to be 47 times lower than that of the blastocyst and the amount in the two-cell embryo was 55 times lower than the amount in the blastocyst in this experiment. The changes in U3 snRNA abundance during early mouse development were verified by RNAse protection assays. When hybrids were formed between aliquots of mouse oocyte and embryo RNA preparations and a radiolabeled riboprobe containing the complement to an entire Ulb-2 gene, the pattern of RNAse-resistant fragments resolved by gel electrophoresis for Ula and Ulb RNA species (Fig. 4B) was similar to that reported previously (Lobo et al., 1988). In brief, Ula is more abundant than Ulb in oocytes and two-cell embryos but the pattern is virtually identical at both stages. Between the two-cell and blastocyst stages, the total amount of Ul RNA increases roughly lo-fold. On the basis of densitometric measurements of the signals for the various Ula and Ulb fragments, corrected for oocyte or embryo number, the amount of Ulb RNA increases about 35-fold in abundance, whereas Ula RNA increases only 6-fold over the same developmental series. A rat U3 antisense riboprobe was hybridized to aliquots of the same RNA samples used for the Ul RNAse protection assays (Fig. 4B). Since the Ul and U3 probes were of identical specific activity and the Ul and U3 bands were of similar size, direct comparison of the RNAse-protected fragments on the gels should be roughly proportional to the number of molecules in

U4, U5, and U6) localization during mouse oocyte maturation, fertilization, and early development. DNA corresponding Sm antibody staining (B, D, F, H, J, L). In the germinal vesicle stage oocytes, snRNP nucleoplasm, with the greatest concentration of antigen surrounding, but excluded from, the nucleolus (A, into punctate foci (C, D) and later relocate throughout the cytoplasm or become extractable. In unfertilized due to extraction (E, F). In early pronuclear stage zygotes (20 hr post-hCG) the enlarging pronuclei bind the throughout the nucleoplasm by the late pronucleate stage. At the mid two-cell stage (44 hr post-hCG) the the nuclei and the second polar body nucleus (I, J). At telophase and prometaphase in chromosomes in an post-hCG; K, L), the antigen is near the poles at telophase and seen as small foci near the prometaphase detection continues to occur during preimplantation development. (Bar = 10 pm).

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FIG. 3. Time-lapse video microscopy of nucleolus formation. Sperm-oocyte fusion, penetration, and second polar body extrusion occur within 3 hr of insemination (A). Within the next hour numerous small nucleolar bodies appear within the developing pronuclei (B; arrows). Over the next 30 min, several of these small structures fuse with each other to form a single large nucleolus (C-F; arrows point to unifying nucleolar bodies). Time (hr) post-hCG is noted. 160X.

each case. Three U3 RNAse-protected fragments were observed in the embryo RNA samples; the two smaller bands together were 45% as intense as the largest (130 nucleotides) band. These arise from cleavage of the hybrid at mismatches between mouse U3 RNA and the rat U3 gene. From the results of the experiment in Fig. 4B, the following observations can be made: (i) The amount of U3 snRNA in oocytes and two-cell embryos is much lower than that of Ul snRNA. The largest protected fragment for U3 snRNA in oocytes and two-cell embryos is slightly less intense than the Ulb-2 band by itself. (ii) There is a marked increase, on an embryo basis, in the amount of U3 snRNA between the two-cell and blastocyst stages. This increase is greater than the 35-fold increase in Ulb-2 over the same developmental interval. By the blastocyst stage, the total amount of U3 snRNA is roughly one-quarter of the total amount of Ul snRNA. These results are completely consistent with the results of the Northern blot analyses presented in Fig. 4A. A summary of the quantitative estimates of the number of U3 and Ul snRNA molecules present in early mouse embryos is included in Table 1.

DISCUSSION

The number of U3 snRNA molecules in ovulated oocytes and two-cell embryos is lower than the corresponding series of snRNAs (Ul, U2, U4, U5 and U6) involved in pre-mRNA processing by a factor of roughly 20 to 40 (Lobo et ul., 1988; Dean et al., 1989). By comparison of the small amounts of U3 snRNA detected in oocytes and two-cell embryos by Northern blot and nuclease protection experiments with the much larger amounts detected in eight-cell embryos and blastocysts, it is estimated that the U3 snRNA content increases roughly 50-fold during this developmental interval. The increase and accumulation must be due to the transcriptional activation of these genes at the two-cell and later stages. While Ul, U2, U4, U5, and U6 snRNAs are involved with pre-mRNA processing, U3 snRNA appears to be involved with processing events that occur at or beyond the 3’ end of 28 S rRNA (Parker and Steitz, 1987). Although U3 snRNAs are associated with nucleoli (Lischwe et ul., 1985), in situ hybridization could not be conducted on the embryos due to the homology

U3 snf?NA A

E

2c

8c

B

1

2

3

4

E2

2-2

B2

Ul snRNA E

2c

8ci3

1

Ui snRNA

B

2

3

U3

4

snRNA

Ulah

67.

M

12

345678

Frc;. 4. Abundance of U3 RNA in early mouse embryos. (A) Northern blot analysis. Left blots: RNA rvas extracted in the presence of tRNA carrier from large pools of ovulated oocgtes and early embryos and divided into several series of equal aliquots such that each contained RNA from the equivalent of 228 unfertilized oocytes (E), 1% two-cell embryos (2~1, 200 eight-cell embryos (Hc), or 133 blastorysts (Bl. The one series of samples was resolved on formaldehyde-agarose gels along with a standard RNA preparation from embryonal carcinoma cells that contained 15, 30, 45, or 60 pg of sense-strand I73 transcripts (lanes 1 to 4, respectively). The second series was resolved on a parallel gel beside Ul sense-strand standard RNA samples in the amounts of 70, 95, 120, and 145 pg (lanes 1 to 4, respectively). Following transfer to Nytran membranes, the blots were hybridized with [a-““S]UTP labeled anti-sense RNA probes (sp act, 1.3 X lo* or 2.0 X IO” cpm/& for IT3 and Ul RNA, respectively). Following washes, blots were exposed to X-ray film at ~70°C for 30 days in the case of the U3 blot and 12 days for the Ii1 blot. 173 snRNA, right blot: RNA from 1079 unfertilized oocytes (EZ), 1%21 two-cell embryos (2-Z), and 50 blastocysts (B2) was Northern blotted as above and hybridized with a I73 antisense RNA transcript labeled in the presence of [W ,“P]UTP to a specific activity of 1.5 X lO’cpm/pg. The washed blot was exposed to X-ray film at -70°C for 6 days. (BJ Nuclease protection assay. RNA \vas prepared in the presence of 10 kg of yeast tRNA carrier from 305 unfertilized oocytes, 313 two-cell embryos, 177

between U3 snRNA and rRNA which resulted in high background in all samples. The large increase in U3 snRNA content after the two-cell stage correlates with the acquisition of the capacity for rRNA synthesis and processing beginning in the late two-cell embryo. While the incorporation of radiolabeled adenosine into heterodisperse RNA is detectable in the zygote and early two-cell stage, there is no detectable synthesis of rRNA until the late two-cell stage (Knowland and Graham, 1972; Clegg and Piko, 1983). From the two-cell stage to the blastocyst stage there is a significant increase in rRNA content and ribosome numbers due to transcription and processing of ribosomal precursors (Piko and Clegg, 1982). Findings from ultrastructural and autoradiographic studies of nucleoli and rRNA synthesis in early cleavage stages of mouse embryos support the biochemical data (Hillman and Tasca, 1969). The multiple nucleoli of the early two-cell embryo are composed of only fibrils. These nucleoli advance in development during the two-cell stage so that prior to second cleavage, some of the nucleoli reticulate and become composed of a dense fibrillar core and a fibrillogranular cortex (definitive nucleoli). The amount of [S”H]uridine incorporation increases in direct proportion to the amount of granularity of the nucleoli (Hillman and Tasca, 1969). In four-cell embryos, at least two of the nucleoli are reticulated while others remain as nonreticulated bodies. By the eight-cell and later stages, usually only two nucleoli are found in each nucleus and these have the ultrastructural characteris-

eight-cell embryos, and 100 blastocysts. Radiolabeled antisense transcripts of 394 nucleotides in length for mouse IJlh-2 snRNA and 340 nucleotidcs in length for rat 173 snRNA were generated in the presence of [@P]UTP and hybridized in excess to aliquots of the mouse oocyte and embryo RNA samples. IJnhybridized tnolecules were digested with ribonuclease and the RN&e-resistant fragments for both Ul and 173 probes were resolved on acrylamide gels along with DNA markers (lane MJ produced by the digestion of pUCY with H/,(111. For the IJlb-Z probe, RNAse-resistant fragments produced following hgbridization to embryo RNA are 165 nucleotides long (resulting from full-length protection by Ulb-2 and IJlb-6 snRNAJ, 135 nuclcotidcs long (IJlb-v due to hybridization with Ulb-5 snRNA which differs from IJlb-2 at nucleotides 32 and 33), and ‘70 to 80 nucleotides long as a result of hybridization by IJla snRNA molecules that differ from IJlb-2 1)s several mismatches near the center of the molecule (see Lobo et ctb. 1988). Three fragments of the rat IT3 probe are protected by mouse embryo RNA samples. The same three RNAsc-resistant IJ3 bands were also produced in the same proportions by hybridization with mouse L-cell RNA (data not shown). The band labeled b in the I73 snRNA assay is a background band that is also generated by the tRNA carrier. Lanes 1 to 8 contain results of the nuclease protection experiment with aliquots of RNA derived from the equivalent number of oocytes or embryos listed. Lane 1, 76 unfertilized oocytes; lane d, 78 two-cell embryos; lane 3, 39 eight-cell embryos: lane -1, 25 hlastocgsts; lane 5, 152 unfertilized oocytes; lane 6, 156 two-cell embryos; lane 7,39 eight-cell embryos; and lane 8, 25 blastocysts.

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DEVELOPMENTAL BIOLOGY

AMOUNTS

TABLE 1 OF U3 snRNA IN EARLY

MO~ISE Molecules per embryo

Developmental Ovulated Two-cell Eight-cell Blastocyst

stage oocyte

EMBRYOS of RNA (X10- “)

Ul”

U3”

9.7 8.1 28.8 94.3

0.5 0.4 5.3 27.0

” Data from the average of six separate RNA preparations including those reported in Lobo et al. (1988). “The blastocyst value is the average of five RNA preparations resolved on Northern blots in relation to U3 standards as shown in Fig. 4A. The values for the other stages were calculated relative to the blastocyst on the basis of Northern blots and nuclease protection assays on four independent preparations. The blastocyst contained 56 times as much U3 snRNA as the oocyte (range of values, 45-73), 70 times as much as the two-cell embryo (range of values, 55-117) and 5.1 times that of the eight-cell embryo (range of values, 4.2-8.3).

tics of definitive nucleoli. The change in the number of definitive nucleoli and the increase in the amount of U3 snRNA is correlated with the number of nucleolar organizer regions that are argyrophilic, as a single nucleolar organizer region is argyrophilic at the second mitosis and this number progressively increases until after the eight-cell stage, where all six nucleolar organizer regions are argyrophilic (Engel et al., 1977; Hansmann et ah, 1978). Thus U3 snRNA accumulation is directly correlated with biochemical and morphological changes indicative of rRNA synthesis. Immunolocalizations of U3 and non-U3 snRNP show different patterns of spatial segregation in interphase nuclei and a cell-cycle dependent localization. The non-U3 antigens display a pattern similar to that of Ul snRNPs (Lobo et ah, 1988; Nash et al., 1987). Verification of the localization of U3 antigens to prenucleoli granules and nucleoli has been demonstrated with video microscopy and anti-nucleolar antibodies directed toward fibrillarin. During meiosis and mitosis nucleolar bodies break down and U3 antigens disperse into the cytoplasm and become extractable. During nuclear envelope reformation, nucleoli reform and U3 snRNPs become nonextractable within the nucleolus. These cell-cycle dependent events are in agreement with the behavior of U3 snRNPs in other cell lines (Maul and Yasuda, 1989) and of non-U3 snRNPs (Verihejm et al, 1986; Lobo et ab, 1988). The detection of U3 snRNPs during the pronuclear stage and early two-cell stage suggests that maternally derived snRNPs are carried over from the oocyte to the early embryo, as new transcription is unlikely at this stage. In fact RNA synthesis is not a prerequisite for the morphological reformation of nu-

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cleoli after mitosis (Semeshin et al., 1975), but nucleolar reformation proceeds at a slower rate without RNA synthesis. The data presented here add to the present knowledge regarding the transition from maternal control to zygotic control during mouse embryogenesis. During the one-cell and early two-cell stages the developing embryo relies upon maternally derived U3 snRNA and snRNPs. At the late two-cell or four-cell stage the embryo begins producing its own snRNAs (including U3 snRNA) and the amounts of all the snRNAs increase significantly by the blastocyst stage. The source of the associated proteins in the snRNPs is not known, but they are probably produced by the embryo after the two-cell stage. It is likely that during the two-cell to four-cell stage both maternally and paternally derived U3 snRNAs are used for the processing of rRNA. The antigenic localization of U3 snRNPs coincides with the formation of prenucleolar swelling and definitive nucleoli of the developing embryo. The nucleoli are the sites of 28 S rRNA processing and U3 snRNA accumulation during early embryo development; their appearance is correlated with morphological and biochemical changes associated with rRNA production. A major increase in U3 snRNA accumulation is observed after the two-cell stage when the transition from maternal to zygotic control of development occurs in this mammal. This work was supported by grants from the NIH to G.S. (HD 22902) and W.F.M. (GM 27789) and from the Medical Research Council of Canada (MT4854) to G.A.S. The Integrated Microscopy Resource in Madison is supported as an NIH Biomedical Research Technology Resource (RR5’70).

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DEAN, W.L.,SEUFERT, A.C., SCHIJLTZ,G. A.,PRATHER,R.,SIMERLY, C., SCHATTEN, G., PILCH, D. R., and MARZLUFF, W. F. (1989). The small nuclear RNAs for pre-mRNA splicing are coordinately regulated during oocyte maturation and early embryogenesis in the mouse. Development 106,325-334. ENGEL, W., ZENZER, M. T., and SCHMID, M. (1977). Activation of mouse ribosomal RNA genes at the 2-cell stage. Hum. Genet. 38,57-63. FOUR~ROY, J. L. (1982). RNA synthesis in immature mouse oocyte development. J. Exp. Zool. 219, 257-266. HANSMANN, I., GEBAUER, J., BIHL, L., and GRIMM, T. (1978). Onset of nucleolus organizer activity in early mouse embryogenesis and evidence for its regulation. Exp. Cell Res. 114,263-268. HILLMAN, N., and TASCA, R. J. (1969). Ultrastructural and autoradiographic studies of mouse cleavage stages. Amer. J. Anat. 126, 151-173. KAPLAN, G., ABRE~J, S. L., and BACHVAROVA, R. (1982). rRNA accu-

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ET AL.

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mulation and protein synthet.ic patterns in growing mouse oocytes. J. Exp. 2001. 220, 361-3’70. KNOWLAND, J., and GRAHAM, C. (1972). RNA synthesis at the two-cell stage of mouse development. J. Embryol. Exp. Morphol. 27,167-176. LISCH~E, M. A., OCHS, R. L., REDL)Y, R., COOK, R. G., YEOMAN, L. C., TAN, E. M., REICHLIN, M., and BUSCH, H. (1985). Purification and partial characterization of a nucleolar scleroderma antigen (M, = 34,000; p1 8.5) rich in No, No-dimethylarginine. J. Biol. Chmn. 260, 14,304-14,310. LOBO, S. M., MARZLIJFF, W. F., SEIJFERT, A. C., DEAN, W. L., S(‘HIJLTZ, G. A., SIMBRLY, C., and SCHATTEN, G. (1988). Localization and expression of IJl RNA in early mouse embryo development. De?: Riol. 127,349-361. MANIATIS, T., and REED, R. (1987). The role of small nuclear ribonucleoprotein particles in pre-mRNA splicing. Nuture (London) 325, 673-678. MOOKE, G. P. M., LINTERN-MOORE, S., PETERS, H., and FABER, M. (1974). RNA synthesis in the mouse oocyte. J. Cell Biol. 60,416-422. NASH, M. A., KOZAK, SCFIATTFN, G., and

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and embryonic 1133-1142. Oc~ls, R. L., LIS~H~E, M. A., SPOHN, W. H., and Buscri, H. (1985). Fibrillarin: A new protein of the nucleolus identified by autoimmune sera. Biol. Cell 54, 123-134. PARKER, K. A., and STEITZ, J. A. (1987). Structural analyses of the

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U3 snRNPs and nucleolar development during oocyte maturation, fertilization and early embryogenesis in the mouse: U3 snRNA and snRNPs are not regulated coordinate with other snRNAs and snRNPs.

U3 small nuclear ribonucleic acids (snRNA) and U3 small nuclear ribonucleoprotein (snRNP), which are thought to be responsible for ribosomal RNA proce...
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