The abundance and localization of snRNAs and snRNPs involved in processing and splicing of pre-mRNA has been studied during early mouse embryogenesis. The amount of U1, U2, U4, U5 and U6 RNA remains relatively constant between the postovulatory oocyte and 2-cell stage but increases three- to ten-fold in quantity between the 2-cell and blastocyst stages. Localization was examined by in situ hybridization with Ul, U2 and U6 riboprobes and immunofluorescence microscopy using a monoclonal antibody to snRNP antigens. The snRNAs and snRNPs are primarily localized to the germinal vesicle in the preovulatory oocyte but are released and diluted within the cytoplasm of the oocyte during germinal vesicle breakdown and meiotic maturation. They subsequently relocalize to both pronuclei following fertilization and the nuclei of the 2-cell embryo following the first cleavage division. Since the amount of snRNA is constant during the first cleavage, the small amount of pre-mRNA that is synthesized at the time of transcriptional activation in the 2-cell embryo may be spliced and processed by snRNPs of maternal origin.

The process of oocyte growth is characterized by the active synthesis and accumulation of RNA and other macromolecules (R. M. Schultz, 1986). At the time of meiotic maturation, the oocyte undergoes breakdown of the germinal vesicle and a period of transcriptional inactivity follows as cellular and chromosomal changes occur in relation to extrusion of the first polar body and progression to an arrested state at metaphase II of meiosis. Following fertilization, meiosis is completed, pronuclei form and the first cleavage of the mouse embryo occurs about 20 h after the time of sperm entry. Many of the protein synthetic and cellular events related to the first cleavage can be mediated by maternally inherited mRNA molecules since they occur in fertilized eggs treated with the transcriptional inhibitor, a-amanitin (Braude et al. 1979; Flach et al. 1982; Howlett & Bolton, 1985). Development beyond the 2-cell stage, however, is dependent upon transcription from the zygote genome (G. A. Schultz, 1986).

A program for maternal mRNA degradation appears to also be triggered during the period of meiotic maturation (Bachvarova et al. 1985). By the 2-cell stage, the majority of the maternal poly (A)+ RNA molecules (Piko & Clegg, 1982) and a number of specific mRNAs including those coding for histone (Graves et al. 1985), actin (Giebelhaus et al. 1985; Bachvarova et al. 1985), zona pellucida (Philpott et al. 1987), c-mos (Mutter & Wolgemuth, 1987) and tubulin (Paynton et al. 1988) proteins are markedly reduced in abundance when compared to the unfertilized oocyte. At the same time, there is a resumption of transcriptional activity. The synthesis of all major classes of RNA has been initiated in the 2-cell embryo (Clegg & Piko, 1983) and paternally derived gene products are detectable by this stage (Sawicki et al. 1981). Microinjection experiments using plasmids containing the herpes simplex and chicken thymidine kinase genes have further demonstrated the capacity for transcription, post-transcriptional processing and translation of newly synthesized mRNAs in the 2-cell embryo (Brinster et al. 1982; Chen et al. 1986).

The cellular machinery that mediates mRNA processing is composed of snRNA molecules which are found in ribonucleoprotein complexes (snRNPs) in the cell nucleus (for review, see Maniatis & Reed, 1987). In a previous study, we have shown that U1 RNA, which is involved in recognition of the 5’ exon-intron splice junction in pre-mRNA (Mount et al. 1983), is not degraded along with mRNA during the transition from maternal to zygote genome control between the unfertilized oocyte and 2-cell stage (Lobo et al. 1988). Rather, U1 snRNAs and U1 snRNPs have been shown, by in situ hybridization and immunofluorescence microscopy, to be released into the cytoplasm of the unfertilized egg during germinal vesicle breakdown and meiotic maturation and subsequently to relocalize to pronuclei following fertilization (Lobo et al. 1988). Since transcriptional activity is negligible prior to the 2-cell stage and since U1 snRNA is constant during this developmental period, the small amount of pre-mRNA that is synthesized during transcriptional activation of the zygote genome is possibly processed by U1 snRNA of maternal origin. After the 2-cell stage, new U1 snRNA is also synthesized and accumulated (Lobo et al. 1988) along with the general increase in RNA synthetic activity and RNA content that occurs from the 2-cell to blastocyst stage (Piko & Clegg, 1982).

In this communication, we have extended our studies to include all of the major snRNAs and snRNPs (U1, U2, U4, U5 and U6) involved in pre-mRNA splicing (Maniatis & Reed, 1987). There are substantial amounts of ‘U’ RNAs present in the oocyte, similar in amount to those found in somatic cells, and these concentrations are maintained through fertilization and cleavage to the 2-cell stage. The localization pattern from germinal vesicle to oocyte cytoplasm to pronuclei during ovulation and fertilization is similar for the entire complex.

Animals and embryo recovery

Random-bred Swiss albino mice (Charles River Breeding Laboratories) were used throughout the study. Procedures for superovulation, collection of ovarian oocytes with intact germinal vesicles, recovery of ovulated oocytes and recovery of preimplantation-stage embryos are exactly as described in Lobo et al. 1988. Injection of human chorionic gonadotrophin (hCG) was used to induce ovulation. Ovulated oocytes were collected at 14–16 h post-hCG, pronuclear zygotes at 20 h post-hCG, 2-cell embryos at 44 to 46 h post-hCG, 8-cell embryos at 68 to 70 h post-hCG and early blastocysts (about 32 cells) at 92 to 96h post-hCG.

RNA extraction and Northern blots

Total RNA was extracted from oocytes and embryos in the presence of 10 gg of yeast tRNA (nuclease-free, Bethesda Research Labs) because this carrier lacks sequences which hybridize to mammalian ‘U’ snRNA probes (Lobo et al. 1988). Procedures for resolution of RNAs in formaldehydeagarose (1·5%) gels, transfer to Nytran (Schleicher and Schull) membranes, hybridization and washes are as described previously (Lobo et al. 1988).

Probes for SnRNA detection

For U1 snRNA assays, a 394 base pair (SstII/EcoRI) fragment of mouse genomic DNA containing the entire 165 base pair region of a U1b gene (Marzluff et al. 1983) was inserted into the multiple-cloning site of Sp64 and Sp65 plasmids. Antisense riboprobes were generated from Sp65 following linearization with Hindlll while control (sense-strand) riboprobes for in situ hybridization reactions were transcribed after linearizing Sp64 with EcoRl.

For U2 snRNA measurements, a 300 base pair genomic fragment containing a 188 base pair mouse U2 gene (Nojima & Kornberg, 1983) was cloned into pGEM-1. Linearization of the pGEM-1 recombinant plasmid with EcoRl produced an antisense riboprobe upon transcription with Sp6 polymerase and the sense-strand (control) riboprobe was produced following linearization with Hindi 11 and transcription with T7 polymerase.

A mouse U4 cloned DNA sequence was not available to us. Hence, a synthetic 50 nucleotide oligomer complementary to nucleotides 34 to 83 of the 145 full-length gene sequence published by Reddy (1984) was obtained from the Regional DNA Synthesis Laboratory, Department of Medical Biochemistry, University of Calgary. Similarly, a 50 nucleotide synthetic oligomer complimentary to nucleotides 34 to 83 of the 117 base pair mouse U5 sequence (Reddy, 1984) was also produced.

U6 SnRNAs were analyzed by use of a 250 base pair fragment (BamHI/EcoRI) of Drosophila genomic DNA containing the entire 107 nucleotide sequence of a U6 gene (Das et al. 1987) that was cloned into Sp64 and Sp65. Antisense riboprobes were transcribed in vitro using Sp6 polymerase following linearization with Hindlll and sensestrand riboprobes were generated from Sp64 after linearization with EcoRl. For some of the Northern blots, a synthetic 49 nucleotide oligomer complimentary to nucleotides 32 to 81 of mouse U6 RNA was used as a probe.

To monitor recovery and transfer efficiency of mouse oocyte and embryo RNA in Northern blot experiments, a 1-4 kb BamHI fragment from a human 28S rRNA gene (Gonzalez et al. 1985) was subcloned into pBR322 and used to generate labelled probes to detect mouse 28S rRNA sequences.

Radiolabelled riboprobes for Northern blots, in situ hybridization and RNAse protection experiments were synthesized using the appropriate polymerase and linearized plasmid in the presence of either [α-32P]UTP (800 Ci mmol-1, NEN Dupont) or [α-35S]UTP (1300 Ci mmol-1, NEN Dupont) exactly as described previously (Lobo et al. 1988). Synthetic deoxynucleotide oligomers were 5’-end labelled using polynucleotide kinase and [γ-32P]ATP (3000 Ci mmol-1, NEN Dupont).

In situ hybridization

Procedures used for fixation, embedding, hybridization, posthybridization washes and autoradiography were exactly as described previously (Lobo et al. 1988).

Immunofluorescence microscopy

Y12, a mouse monoclonal antibody that is homologous to anti-Sm antibodies and recognizes U1, U2, U4, U5 and U6 snRNPs (Lerner et al. 1981), was generously donated by Dr T. Martin, Department of Cell Biology, University of Chicago and a sample of an international reference human anti-Sm serum (Tan et al. 1982) was provided as a gift by Dr M. Fritzler, Department of Medicine and Medical Biochemistry, University of Calgary. The supernatant of Y12 was diluted 1:2 in phosphate-buffered saline (PBS) whereas the human anti-Sm serum was diluted 1:10 prior to experimental use. All other procedures for fixation and permeabilization of mouse oocytes and embryos, application of primary and secondary antibodies, observation of stained material and photography were conducted as described by Lobo et al. (1988).

Changes in snRNA abundance during early development

The amounts of U1, U2, U4, U5 and U6 snRNAs within RNA preparations from mouse oocytes, 2-cell embryos, 8-cell embryos and blastocysts were estimated by Northern blot analysis. Fig. 1 is a composite photograph of parts of several blots (each hybridized with a different probe) that all contain identical samples of a single series of RNA preparations. The loading in each case represents the amount of RNA derived from the equivalent of 228 unfertilized oocytes, 186 2-cell embryos, 200 8-cell embryos and 133 early blastocysts. The ribosomal RNA content of mouse oocytes and early embryos is reported to be 0·22, 0·17, 0·40 and 1·00ng per mouse oocyte, 2-cell embryo, 8-cell embryo and early blastocyst, respectively (Piko & Clegg, 1982). Using these values, the comparative rRNA content expected within the set of RNA samples we have used, when corrected for embryo number, is calculated to be 1·00 oocyte: 0·63 2-cell: 1·59 8-cell: 2·65 blastocyst. When one blot was hybridized with a 28S rRNA probe (Fig. 1) and scanned in a densitometer, the relative hybridization intensity observed was 1:00 oocyte: 0·72 2-cell: 1·44 8-cell: 3:11 blastocyst. Tirus, the relative efficiency of RNA extraction, recovery and transfer within this set of RNA preparations was similar, resulting in relative amounts of 28S RNA close to that expected. The relatively higher value for our blastocyst RNA preparations suggests that the embryos in this sample probably contained, on average, slightly more than 32 cells, the cell number upon which Piko & Clegg’s (1982) data is based.

Fig. 1.

Northern blot analysis of U1, U2, U4, U5 and U6 RNA from mouse eggs and early embryos. RNA was extracted in the presence of 10 μg of yeast tRNA carrier from 912 unfertilized oocytes, 744 2-cell embryos, 800 8-cell embryos and 532 blastocysts. Each sample was divided into four equal samples and resolved on 1 · 5 % formaldehyde-agarose gels and transferred to Nytran membranes. Three of the four identical blots were then hybridized with radiolabelled probes for U1, U4 and U5 RNAs, respectively. Following decay to background levels, the U1 blot was rehybridized with a 28S rRNA probe to compare efficiency of extraction and recovery between preparations, the U4 blot was rehybridized with a U2 probe and the U5 blot was rehybridized with a U6 probe. Antisense riboprobes generated in the presence of [α-‘35S]UTP to a specific activity of 2×108ctsmin-1μg-1 were used for U1 and U2 blots with an X-ray film exposure time of 12 days. The U4, U5 and U6 blots were hybridized with synthetic deoxynucleotide polymers (50-mers) 5’end-labelled with [γ-32P]ATP to a specific activity of 1 to 2×107ctsmin-1 an X-ray film exposure time of four to five days was used, lite 28S rRNA probe was labelled by nick-translation in the presence of [α32P]dCTP to a specific activity of 1×107ctsmin-1μg-1 and a very short X-ray film exposure time was utilized.

Fig. 1.

Northern blot analysis of U1, U2, U4, U5 and U6 RNA from mouse eggs and early embryos. RNA was extracted in the presence of 10 μg of yeast tRNA carrier from 912 unfertilized oocytes, 744 2-cell embryos, 800 8-cell embryos and 532 blastocysts. Each sample was divided into four equal samples and resolved on 1 · 5 % formaldehyde-agarose gels and transferred to Nytran membranes. Three of the four identical blots were then hybridized with radiolabelled probes for U1, U4 and U5 RNAs, respectively. Following decay to background levels, the U1 blot was rehybridized with a 28S rRNA probe to compare efficiency of extraction and recovery between preparations, the U4 blot was rehybridized with a U2 probe and the U5 blot was rehybridized with a U6 probe. Antisense riboprobes generated in the presence of [α-‘35S]UTP to a specific activity of 2×108ctsmin-1μg-1 were used for U1 and U2 blots with an X-ray film exposure time of 12 days. The U4, U5 and U6 blots were hybridized with synthetic deoxynucleotide polymers (50-mers) 5’end-labelled with [γ-32P]ATP to a specific activity of 1 to 2×107ctsmin-1 an X-ray film exposure time of four to five days was used, lite 28S rRNA probe was labelled by nick-translation in the presence of [α32P]dCTP to a specific activity of 1×107ctsmin-1μg-1 and a very short X-ray film exposure time was utilized.

A major feature of all the blots in Fig. 1 is that the relative ‘U’ snRNA content, when corrected for embryos numbers, is about the same in the unfertilized oocyte and 2-cell preparations. The amount increases slightly in 8-cell embryos and by the early blastocyst stage, densitométrie measurements (corrected for embryo numbers) demonstrate that the amount is 10·5 times that of the oocyte in the case of U1 RNA, 5T times that of the oocyte for U2 RNA, 3·8 times that of the oocyte for U4 RNA, 5·2 times that of the oocyte for U5 RNA and 3·8 times that of the oocyte for U6 RNA.

To quantify ‘U’ RNA abundance in absolute terms, the ‘U’ RNA content of a nuclear RNA preparation from nullipotent embryonal carcinoma (EC) cells was established by hybridization of radiolabelled antisense riboprobes to slot blots of increasing amounts of EC cell RNA in parallel with known quantities of sense-strand RNA from transcription vectors. An example of the titration of the EC cell RNA preparation for U2 RNA content is shown in panel B, Fig. 2. Similar experiments were conducted for U1 RNA (Lobo et al. 1988) and U6 RNA (data not shown). The EC cell RNA preparation was then used as a standard to compare the intensity of hybridization of ‘U’ probes to RNA from mouse oocytes and early embryos. For U2 RNA, the sample of RNA from 133 blastocysts contains about 120 pg U2 sequence in comparison to the EC cell standard series (panels A and C, Fig. 2). Similar approaches were used to quantify U1 and U6 RNA in oocyte and embryo preparations. The number of U1, U2 and U6 RNA molecules per oocyte or embryo were calculated following correction for efficiency of extraction (estimated to be 69 ± 2 %) and taking into account RNA sizes of 165, 188 and 107 nucleotides, respectively (Table 1).

Table 1.

Absolute amounts of snRNA in mouse embryos

Absolute amounts of snRNA in mouse embryos
Absolute amounts of snRNA in mouse embryos
Fig. 2.

Measurement of the amount of U2 RNA in early mouse embryos. (A) Northern blot analysis of RNA from early mouse embryos and embryonal carcinoma cells. RNA from the equivalent of 228 unfertilized oocytes (E), 186 2-cell embryos (2c), 200 8-cell embryos (8c) and 133 blastocysts (B) was resolved on formaldehyde agarose gels along with increasing amounts of EC cell RNA containing the amount of U2 sequence indicated. Following transfer to Nytran membrane, hybridization with a 35S-labeUed riboprobe was carried out as described in Fig. 1. (B) Slot blot of embryonal carcinoma (EC) cell RNA and U2 sense-strand RNA. RNA from EC cells was applied to Nytran membrane in concentrations ranging from 10 to 110 ng alongside sense-strand U2 RNA, generated by T7 polymerase, in concentrations ranging from 10 to 120 pg. Following hybridization with antisense riboprobe, densitometry was performed and from points of overlap in hybridization intensity from the slot blot, it was determined that 10 ng of EC cell RNA gave the same hybridization signals as 28·5 pg of sense-strand RNA. (C) U2 RNA standard curve Following densitometry of the hybridization signals from the Northern blot in A, a graph was constructed from the EC cell U2 RNA values to measure the comparable U2 RNA content in the RNA sample from 133 blastocysts (arrow). (D) U2 RNAse protection experiments. A 300 nucleotide, 32P-labelled, antisense riboprobe was generated using Sp6 polymerase and hybridized in excess to RNA from mouse embryos and myeloma cells. Unhybridized molecules were digested with ribonuclease and the 188 nucleotide RNAse-resistant duplexes were resolved on acrylamide gels according to procedures described in Lobo et al. (1988). RNA was obtained from 100 unfertilized oocytes (lane E), 50 2-cell embryos (lane 2c), 50 8-cell embryos (8c), and two preparations of 50 blastocysts (Bl and B2). RNA from mouse myeloma cells (0-1, 10 and 10ng, respectively, in lanes M1, M2 and M3) and carrier yeast tRNA (T) were included for comparison.

Fig. 2.

Measurement of the amount of U2 RNA in early mouse embryos. (A) Northern blot analysis of RNA from early mouse embryos and embryonal carcinoma cells. RNA from the equivalent of 228 unfertilized oocytes (E), 186 2-cell embryos (2c), 200 8-cell embryos (8c) and 133 blastocysts (B) was resolved on formaldehyde agarose gels along with increasing amounts of EC cell RNA containing the amount of U2 sequence indicated. Following transfer to Nytran membrane, hybridization with a 35S-labeUed riboprobe was carried out as described in Fig. 1. (B) Slot blot of embryonal carcinoma (EC) cell RNA and U2 sense-strand RNA. RNA from EC cells was applied to Nytran membrane in concentrations ranging from 10 to 110 ng alongside sense-strand U2 RNA, generated by T7 polymerase, in concentrations ranging from 10 to 120 pg. Following hybridization with antisense riboprobe, densitometry was performed and from points of overlap in hybridization intensity from the slot blot, it was determined that 10 ng of EC cell RNA gave the same hybridization signals as 28·5 pg of sense-strand RNA. (C) U2 RNA standard curve Following densitometry of the hybridization signals from the Northern blot in A, a graph was constructed from the EC cell U2 RNA values to measure the comparable U2 RNA content in the RNA sample from 133 blastocysts (arrow). (D) U2 RNAse protection experiments. A 300 nucleotide, 32P-labelled, antisense riboprobe was generated using Sp6 polymerase and hybridized in excess to RNA from mouse embryos and myeloma cells. Unhybridized molecules were digested with ribonuclease and the 188 nucleotide RNAse-resistant duplexes were resolved on acrylamide gels according to procedures described in Lobo et al. (1988). RNA was obtained from 100 unfertilized oocytes (lane E), 50 2-cell embryos (lane 2c), 50 8-cell embryos (8c), and two preparations of 50 blastocysts (Bl and B2). RNA from mouse myeloma cells (0-1, 10 and 10ng, respectively, in lanes M1, M2 and M3) and carrier yeast tRNA (T) were included for comparison.

The number of U2 RNA and U6 RNA molecules in the ovulated oocyte (about 3 × 106 copies) are estimated to be roughly three times that of the U1 RNA molecules (Table 1). The numbers remain relatively constant in 2-cell embryos but copy numbers increase roughly threefold by the blastocyst stage in the case of U2 and U6 RNA and tenfold in the case of U1 such that all are present at a level of roughly l ×107 molecules per embryo or 2·8×105 molecules per cell. These changes in RNA abundance have been verified by nuclease protection experiments for both U1 RNA (Lobo et al. 1988) and U2 RNA (panel D, Fig. 2). Hybridization of excess antisense U2 riboprobe in solution to RNA from oocytes and embryos yields a single full-length RNAse-resistant fragment of 188 nucleotides and suggests that, in contrast to U1 RNA (Lobo et al. 1988), no variants of U2 RNA are expressed during preimplantation mouse development. The amount of U2 RNA in the nuclease protection assay indicates that 100 oocytes have about twice as much U2 RNA as 50 2-cell embryos and that 50 blastocysts contain roughly four times as much U2 RNA as 50 2-cell embryos. The amount of U2 RNA in 50 blastocysts (roughly 1600 cells) is similar to that of 10 ng of myeloma cell RNA (derived from the equivalent of about 500 cells).

Localization of snRNAs during early embryogenesis The intracellular distribution of U1, U2 and U6 RNA in mouse oocytes and early embryos was analyzed by in situ hybridization with [α35S]UTP-labelled antisense riboprobes (Fig. 3 and Fig. 4). Specificity of hybridization was verified by comparing silver grain density in sections hybridized with antisense riboprobes to the low silver grain density obtained in sections hybridized with sense-strand riboprobes under identical experimental conditions (see U6 sense-probe hybridization in column C, Fig. 3). Interestingly, silver grain densities in oocyte and early cleavage stage embryo sections were slightly higher with antisense U2 and U6 riboprobes compared with U1 probes under the same in situ hybridization conditions, an observation that is consistent with the quantitative estimates above.

Fig. 3.

Localization of U1 and U6 snRNA in sections of mouse oocytes and preimplantation embryos by in situ hybridization. Preovulatory oocytes with intact germinal vesicles were recovered from ovaries of females stimulated 44 h earlier with pregnant mare serum gonadotrophin. Unfertilized oocytes were recovered from oviducts of unmated females 14 h after induction of ovulation with hCG. Fertilized eggs with pronuclei were recovered from reproductive tracts of mated females at 20–22 h post-hCG, 2-cell embryos at 44h post-hCG, 8 to 16-cell morulae at 72 to 80 h post-hCG and early blastocysts at 96 h post-hCG. Oocytes or embryos were fixed in Carnoy’s, embedded in paraffin and sectioned at 5 μm thickness. Representative stained samples of each stage are presented in the left column. Samples in the second column of micrographs were hybridized with antisense U6 probe while those in the third column (C) were hybridized with control (sense) U6 probe. The right column presents the dark-field photomicrographs of comparable sections hybridized with an antisense U1 probe. Autoradiographic exposure time was 6 days.

Fig. 3.

Localization of U1 and U6 snRNA in sections of mouse oocytes and preimplantation embryos by in situ hybridization. Preovulatory oocytes with intact germinal vesicles were recovered from ovaries of females stimulated 44 h earlier with pregnant mare serum gonadotrophin. Unfertilized oocytes were recovered from oviducts of unmated females 14 h after induction of ovulation with hCG. Fertilized eggs with pronuclei were recovered from reproductive tracts of mated females at 20–22 h post-hCG, 2-cell embryos at 44h post-hCG, 8 to 16-cell morulae at 72 to 80 h post-hCG and early blastocysts at 96 h post-hCG. Oocytes or embryos were fixed in Carnoy’s, embedded in paraffin and sectioned at 5 μm thickness. Representative stained samples of each stage are presented in the left column. Samples in the second column of micrographs were hybridized with antisense U6 probe while those in the third column (C) were hybridized with control (sense) U6 probe. The right column presents the dark-field photomicrographs of comparable sections hybridized with an antisense U1 probe. Autoradiographic exposure time was 6 days.

Fig. 4.

Localization of U2 snRNA in mouse oocytes and preimplantation-stage embryos. Sections derived from the stages listed in Fig. 3 were hybridized in situ with an antisense U2 riboprobe. Bright-field photomicrographs include: (A) preovulatory oocyte with germinal vesicle; (B) ovulated oocyte; (C) fertilized egg section through one pronucleus; (D) fertilized egg section that does not pass through a pronucleus; (E) fertilized egg with section passing through both pronuclei; (F) 2-cell embryo with section passing through nuclei of each blastomere; (G) 2-cell embryo in which section excludes nuclei; (H) section through five nuclei of an 8-cell embryo; (I) section through the ICM component of a blastocyst, and (J) section through the trophectoderm component of a blastocyst.

Fig. 4.

Localization of U2 snRNA in mouse oocytes and preimplantation-stage embryos. Sections derived from the stages listed in Fig. 3 were hybridized in situ with an antisense U2 riboprobe. Bright-field photomicrographs include: (A) preovulatory oocyte with germinal vesicle; (B) ovulated oocyte; (C) fertilized egg section through one pronucleus; (D) fertilized egg section that does not pass through a pronucleus; (E) fertilized egg with section passing through both pronuclei; (F) 2-cell embryo with section passing through nuclei of each blastomere; (G) 2-cell embryo in which section excludes nuclei; (H) section through five nuclei of an 8-cell embryo; (I) section through the ICM component of a blastocyst, and (J) section through the trophectoderm component of a blastocyst.

The pattern of localization during early development is the same for U1, U2 and U6 RNA (Fig. 3 and Fig. 4). In the preovulatory oocyte, silver grains are primarily localized to the germinal vesicle nucleus. In ovulated oocytes in which germinal vesicle breakdown has occurred, the Ul, U2 and U6 snRNA transcripts are uniformly distributed throughout the cytoplasm. Early postfertilization zygotes contain silver grains that are highly enriched when the section passes through either one (Fig. 3) or both pronuclei (Fig. 4) of a fertilized egg. The preferential nuclear localization of U1, U2 and U6 snRNA is then maintained in 2-cell embryos, 8-cell embryos, morulae and both inner cell mass and trophectoderm cells of the blastocyst (Fig. 3 and Fig. 4). The localization is, indeed, nuclear since sections of pronuclear fertilized eggs or 2-cell embryos that do not pass through the nucleus also exhibit very low silver grain densities that are not detectably different from those obtained with control (sense-strand) riboprobes (compare panels C and D and panels F and G in Fig. 4).

Localization of Sm antigen in snRNPs

The U class of snRNAs is present in ribonucleoprotein particles which are recognized by antibodies to Sm antigen that are present in sera of some patients with the autoimmune disorder systemic lupus erythematosus (Lerner & Steitz, 1979). The distribution of snRNPs in mouse oocytes and early embryos was examined by immunofluorescence microscopy using a mouse monoclonal antibody, Y12, that is homologous to anti-Sm antibodies and with human anti-Sm antibodies (Fig. 5). The two antibody preparations yielded identical results except at the pronuclear zygote stage where the human anti-Sm serum was more effective in detection of snRNPs.

Fig. 5.

Y12 and anti-Sm snRNP immunofluorescent detection in mouse oocytes and preimplantation zygotes. Y12 snRNP antibody staining [Y12; B,D,H,J] or anti-Sm [Sm;F] antibody staining are shown on the right and corresponding Hoechst DNA fluorescent localization [DNA; A,C,E,G,I] is shown on the left. (A,B) An immature oocyte with intact germinal vesicle: Y12 snRNP antibody is localized throughout the nucleoplasm of the germinal vesicle but is excluded from the nucleolus. (C,D) Unfertilized oocyte arrested at second meiotic metaphase. No staining is observed in the meiotic spindle or chromosomes; only faint fluorescence is detected in the cytoplasm of the unfertilized oocyte. (E,F) Pronucleate stage, 20h post-hCG: snRNPs are detected in both pronuclei with an anti-Sm antibody, Y12 does not react with pronuclei. (G,H) 2-cell stage, 44 h post-hCG: At the mid-2-cell stage, Y12 antibody staining is localized throughout the nucleoplasm in both daughter blastomeres. (I,J) Morula stage, 80 h post-hCG: In later preimplantation stages, blastomeres restrict Y12 antigen to each interphase nucleus.

Fig. 5.

Y12 and anti-Sm snRNP immunofluorescent detection in mouse oocytes and preimplantation zygotes. Y12 snRNP antibody staining [Y12; B,D,H,J] or anti-Sm [Sm;F] antibody staining are shown on the right and corresponding Hoechst DNA fluorescent localization [DNA; A,C,E,G,I] is shown on the left. (A,B) An immature oocyte with intact germinal vesicle: Y12 snRNP antibody is localized throughout the nucleoplasm of the germinal vesicle but is excluded from the nucleolus. (C,D) Unfertilized oocyte arrested at second meiotic metaphase. No staining is observed in the meiotic spindle or chromosomes; only faint fluorescence is detected in the cytoplasm of the unfertilized oocyte. (E,F) Pronucleate stage, 20h post-hCG: snRNPs are detected in both pronuclei with an anti-Sm antibody, Y12 does not react with pronuclei. (G,H) 2-cell stage, 44 h post-hCG: At the mid-2-cell stage, Y12 antibody staining is localized throughout the nucleoplasm in both daughter blastomeres. (I,J) Morula stage, 80 h post-hCG: In later preimplantation stages, blastomeres restrict Y12 antigen to each interphase nucleus.

The general pattern of localization of the protein component of snRNPs was similar to that observed by in situ hybridization of the snRNA component of the ribonucleoprotein particles. Y12 and Sm antigens were readily detected in the germinal vesicle of the preovulatory oocyte (Fig. 5A and 5B). In the ovulated oocyte arrested at second metaphase of meiosis, the fluorescence was almost totally lost as the antigen was diluted and distributed throughout the cytoplasm (Fig. 5C and 5D). Following fertilization and development of pronuclei, Sm antigen could be detected in both the male and female pronucleus (Fig. 5E and 5F) and in the mid-2-cell stage, anti-Y12 and anti-Sm antibodies localized to the nucleoplasm of both blastomere nuclei (Fig. 5G and 5H). At the blastocyst stage, the RNP antigen, along with snRNA (Fig. 4), was primarily localized to the nucleus of each cell (Fig. 51 and 5J).

The transition from control by maternal mRNA molecules accumulated during oogenesis to newly synthesized transcripts from the zygote genome occurs during the first cleavage of the developing mouse embryo (G. A. Schultz, 1986). By the 2-cell stage, the early embryo can synthesize and process messenger RNA precursors into functional templates for protein synthesis. In a previous study, we analyzed U1 snRNA and snRNP localization and expression to obtain preliminary information about the mRNA processing machinery during this critical phase of early mouse development (Lobo et al. 1988). In this report, we have extended these studies to the additional components of the splicesome complex to demonstrate that other snRNAs are coordinately expressed with U1 RNA during early development.

From a quantitative point of view, the pattern of change in abundance of U1, U2, U4, U5 and U6 RNAs is very similar between the egg to blastocyst stage. The amount of each remains more- or-less constant during the period of transcriptional inactivity that exists prior to fertilization of the ovulated oocyte until the completion of the first cleavage. That is, no components of the maternal snRNA pool are degraded during this interval where maternal mRNA levels decrease markedly (Piko & Clegg, 1982; Giebelhaus et al. 1985; Graves et al. 1985). Prior to the 8-cell stage, synthesis of all five snRNAs must be initiated to account for the increased abundance that begins to be detectable at this stage and leads to marked increases, on an embryo basis, by the blastocyst stage. Interestingly, U1 RNA content increases about tenfold from the egg to blastocyst stage whereas the abundance of U2, U4, U5 and U6 RNA molecules increases only threefold to fivefold over the same developmental interval. On a cellular basis, the unfertilized oocyte contains about 1 × 106 molecules of U1 RNA and 3 × 106 molecules of U2 and U6 RNA. By the blastocyst stage, the number of molecules per cell is about 3 ·0 × 105 in each case. This is similar to the number of snRNA molecules in somatic cells that contain about one third to one half the number observed in cultured myeloma cells (Lobo et al. 1988) and HeLa cells (Zieve & Penman, 1976). We did not have appropriate recombinant plasmids to measure U4 or U5 RNA molecules in relation to standards as was possible in the case of U1, U2 and U6 RNA. Nonetheless, the intensity of hybridization of 5’end-labelled synthetic deoxynucleotide (50-mer) probes to a standardized set of Northern blots was very comparable for U4, U5 and U6 sequences. It appears, therefore, that U4 and U5 RN A concentrations are closely similar to those reported in Table 1 for U6 RNA.

In terms of localization, in situ hybridization has shown that U1, U2 and U6 snRNAs are predominantly confined to the transcriptionally active germinal vesicle of the preovulatory oocyte but are then released into the oocyte cytoplasm during germinal vesicle breakdown that accompanies meiotic maturation. A parallel loss of immunofluorescent staining intensity occurs as antibodies recognizing the protein component of snRNPs localize in a diffuse and diluted manner throughout the oocyte cytoplasm. U1, U2 and U6 snRNAs relocalize to newly formed pronuclei in the fertilized zygote and the nuclei of the first two blastomeres following the first cleavage. Comparable relocation of RNP antigens is also observed in both pronuclei of the fertilized oocyte and both nuclei of the 2-cell embryo by immunofluorescent microscopy. The nuclear location of snRNAs and snRNP antigens is maintained in later embryonic stages.

The relocation of snRNAs and snRNPs into nuclei by the 2-cell stage of mouse embryogenesis coincides with the timing of transcriptional activation of the zygote genome. Large amounts of U1 snRNAs have also been demonstrated within the germinal vesicle of Xenopus oocytes and these are also dispersed during germinal vesicle breakdown (Forbes et al. 1983; Zeller et al. 1983; Fritz et al. 1984). At the time of transcriptional activation at the mid-blastula transition, new U1 and U4 embryonic variants are synthesized (Forbes et al. 1984; Lund & Dahlberg, 1987). In the sea urchin oocyte, U1 RNA is also accumulated in the germinal vesicle of the oocyte but is not detectable within nuclei of the early embryo until it first reappears, apparently due to new synthesis, in micromere nuclei at the 16-cell stage and subsequently in all nuclei of the embryo (Nash et al. 1987). Thus, each developmental system appears to reflect a pattern of snRNA expression and localization that may be related to the period of maternal control of development. In this regard, it will be interesting to examine whether snRNAs and snRNPs of maternal origin are localized and expressed differently in other mammalian species like the rabbit (Manes, 1973) or the sheep (Crosby et al. 1988) where the period of maternal control (with delayed activation of zygotic transcription) extends to the third or fourth cleavage.

This work was supported by grants from the NIH to W.F.M. (GM 27789) and G.S. (HD 12913 and HD 22902), and by a grant from the Medical Research Council of Canada (MT4854) to G.A.S.; A.S. and W.L.D. received studentships from the Alberta Heritage Foundation for Medical Research. The Integrated Microscopy Resource in Madison is supported as a NIH Biomedical Research Technology Resource (RR570).

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