Proc. Natl. Acad. Sci. USAVol. 89, pp. 10051-10055, November 1992Developmental Biology

Antisense c-myc effects on preimplantation mouseembryo developmentBIBHASH C. PARIA*t, SUDHANSU K. DEY*t, AND GLEN K. ANDREWSt§Departments of *Obstetrics-Gynecology, tPhysiology, and tBiochemistry and Molecular Biology, Ralph L. Smith Research Center, University of KansasMedical Center, Kansas City, KS 66103

Communicated by Clement L. Markert, July 6, 1992

ABSTRACT Antisense DNA inhibition of gene expressionwas explored as an approach toward elucidating mehanismsregulating development of preimplantation mammalian em-bryos. Specifically, a role for the c-myc protooncogene wasexamined. Detection of c-myc mRNA and immunoreactivenuclear c-myc protein in preimplantation mouse embryos at theeight-cell/morula and blastocyst stages suggested that thisDNA-binding protein could be important during early embryo-genesis. The effects of c-myc oligodeoxyribonucleotides (oligos)on the in vitro development of two-cell mouse embryos wereexamined. Embryos cultured in medium containg an unmod-ified (phosphodiester) antisense c-myc oligo complementary tothe translation initiation codon and spanning the first sevencodons exhibited a dose-dependent arrest at the eight-cell/morula stage. At lower concentrations (7.5 FsM) thisinhibitory effect was specific to the antisense oligo and did notoccur with the sense-strand complement or with duplexes oftheantisense and sense oligos. However, at 4-fold higher concen-trations of DNA (30 FM), all unmdified c-myc oligos wereembryotoxic, causing embryos to arrest at the two-cell tofour-cell stages. In contrast, almost all (98%) two-cell embryoscultured with a modified (chimeric phosphorothioate/phos-phodiester) antisense c-myc oligo (7.5 pM) exhibited develop-mental arrest at the eight-cell/morula stage, whereas nodevelopmental arrest occurred following incubation with hihconcentrations of the modified sense complement (30 FM).Culture of freshly recovered eight-cell embryos with antisenec-myc led to the absence of c-myc protein but no change inepidermal growth factor receptor in those embryos that devel-oped a blastocoel. These effects on c-myc were specific for theantisense oligo. These results suggest that c-myc fmctionbecomes particularly critical for preimplantation mouse em-bryos at the eight-cell/morula stage of development and es-tablish that antisense DNA can be successfully applied as anapproach toward elucidating the roles of specific genes inpreimplantation mammalian embryo development.

The fertilized mammalian egg, after completion of severalcleavages, differentiates into a blastocyst. The blastocyst iscomposed of the inner cell mass, which gives rise to theembryo proper and certain extraembryonic membranes, andthe trophectoderm, which participates in the formation ofextraembryonic structures (placenta and parietal yolk sac).Although activation of transcription of the embryonic ge-nome occurs at the two-cell stage in the mouse (1, 2), ourknowledge of genes that are crucial for preimplantationembryo development is meager. In this study, the role ofc-myc in preimplantation mouse embryo development wasexamined. The c-myc protooncogene is the cellular homologof the viral v-myc oncogene and is a member ofa gene familythat includes L-myc and N-myc (3-7). c-myc is a nuclearDNA-binding phosphoprotein that is highly conserved

throughout evolution. It is now thought that biological func-tions and sequence-specific DNA-binding activity of c-mycare executed by dimerization with another protein called Max(8-10). The expression of c-myc is positively correlated withDNA synthesis and cell proliferation (8-10) and is rapidlyinduced in various cell types after stimulation with mitogen(11). A direct role of c-myc in the cell cycle is suggested bythe observation that quiescent cells enter the cell cycle anddivide after microinjection of c-myc protein or transfectionwith the c-myc gene (12, 13). Because of the association ofc-myc with cell cycle in cultured cells (14, 15) and thesuggested role of this protein in cell division and differenti-ation during postimplantation mammalian development (16-19), this protein could be important for cleavage and/ordifferentiation during early mammalian embryogenesis.

Definitive proof for a role of c-myc in preimplantationembryo development requires examination of the effects ofselective ablation of expression of this gene. Because of theextremely short half-life ofc-mycmRNA and protein (20-22),the use of an antisense oligodeoxyribonucleotide (oligo) toc-myc mRNA to block its translation appeared an attractiveapproach toward elucidating the role ofthis protooncogene inpreimplantation embryo development. Since the discovery ofantisense RNAs as natural suppressors of prokaryotic genes(23-27), the successful use of antisense RNA/DNA to ma-nipulate expression ofendogenous and exogenous eukaryoticgenes has been reported (28-34). Furthermore, several lab-oratories have reported inhibition of cell proliferation result-ing from antisense blockage of expression of c-myc duringculture in medium containing antisense oligo (35-38).

MATERIALS AND METHODSMaterials. HPLC-purified modified and unmodified sense

and antisense oligos were purchased from Genosys, Hous-ton. Rabbit polyclonal antibodies to human c-myc werekindly provided by Rosemary Watt (Smith Kline & French).Crystalline bovine serum albumin was purchased from Sigma(A-4378). DNA ladder was obtained from Bethesda ResearchLaboratories. All other reagents were purchased from Sigma,Fisher, or Mallinckrodt.

Animals. Female mice (20-25 g) of Charles River strain(CD-i) were mated with the males of the same strain. Themorning of finding the vaginal plug was designated as day 1of pregnancy.Reverse Transcriptase Polymerase Chain Reaction (RT-

PCR). Amplification and detection of c-myc mRNA in pre-implantation mouse embryos were achieved by RT-PCR.Embryos (about 80 per group) were collected from day 1-4pregnant mice. Due to asynchronous embryonic develop-

Abbreviations: oligo, oligodeoxyribonucleotide; EGF-R, epidermalgrowth factor receptor; RT-PCR, reverse transcriptase polymerasechain reaction.§To whom reprint requests should be addressed at: Department ofBiochemistry and Molecular Biology, University ofKansas MedicalCenter, 39th and Rainbow Blvd., Kansas City, KS.

10051

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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ment, a mixture of four-cell and eight-cell embryos wasobtained on day 3 and late morulae and blastocysts on day 4.Embryos were washed several times to avoid any contami-nation with maternal cells and quick frozen in a small volume(5 LI) of culture medium in the bottom of a 400-.lI microcen-trifuge tube. After addition of Escherichia coli rRNA (20 ,ug)to the tube, total RNA was extracted from these embryosusing sodium dodecyl sulfate/phenol/chloroform buffers(39). c-myc mRNA was reverse transcribed and amplified byPCR as described (39, 40). Oligos (21 bases long) weresynthesized based on the sequence of the mouse c-myc gene(41) as follows:

P1 5'-CCTCTTCTCCACAGACACCAC-3';P2 and P2S 5'-ATGCCCCTCAACGTGAACTTC-3l;P3 5'-CCCTATTTCATCTGCGACGAG-3'.

The antisense oligo termed P1 was complementary to thec-myc gene beginning at base 53 relative to the start of exon 3(Fig. la). P1 served as a primer for reverse transcription ofc-mycmRNA and as the 3' primer during PCR. The sense oligostermed P2 and P2s began at the c-myc translation initiationcodon in exon 2 and spanned the first seven codons (Fig. la).P2 served as the 5' primer during PCR. The sense oligo termedP3 began at codon 21 in exon 2 and was thus internal to the PCRprimers (Fig. la). P3 was 5' end-labeled with 32p using T4kinase, and the labeled primer was used to detect c-myc PCR

a0 1000 2000 3000 4000 5000 6000 bp

c--myc gene

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Exon 1 Exon 2 Exon 3

P1

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Pregnancy

FIG. 1. RT-PCR, amplification, and detection of c-myc mRNA inpreimplantation mouse embryos on days 1-4 of pregnancy. About 80embryos per group were used for each day of pregnancy. (a)Structure of the mouse c-myc gene indicating the locations of theoligo primers (P1.3) used for RT-PCR and/or in embryo cultureexperiments. bp, Base pairs. (b) Southern blot detection of RT-PCRproducts specific for amplification of c-myc mRNA in preimplanta-tion embryos obtained on the indicated days of pregnancy. c-myctranscripts were reverse transcribed into cDNA and then amplifiedusing PCR (45 cycles). RT-PCR was predicted to amplify a 398-bpfragment of c-myc mRNA. Control PCR reactions containing RNAthat had not been reverse transcribed were performed in parallel andyielded negative results. The control (lane C) represents day 4embryo RNA. RT-PCR products were separated by 2% agarose gelelectrophoresis, Southern blotted to nylon membranes, and hybrid-ized to end-labeled P3. This probe is complementary to an internalregion of the predicted c-myc RT-PCR product. Hybrids weredetected by autoradiography.

products by Southern blot hybridization. c-myc transcriptswere reverse transcribed into cDNA and then amplified usingPCR (45 cycles) as described by Andrews et al. (39). Theannealing temperature during PCR was 600C. Control PCRreactions containingRNAthat had not been reverse transcribedwere performed in parallel and yielded negative results. RT-PCR products, equivalent to that obtained from about oneembryo, based on the original sample size, were separated by2% agarose gel electrophoresis, Southern blotted to nylonmembranes, and hybridized at 500C to end-labeled P3 as de-scribed (42). Hybrids were detected by autoradiography. Sizesof RT-PCR products were estimated by comparison with a123-bp DNA ladder. The predicted size ofthe RT-PCR productof c-myc mRNA was 398 bp. This experiment was repeatedtwice using independent isolates of preimplantation embryoRNA, and similar results were obtained.

P2 and its complementary antisense oligo (sequence notshown) were unmodified, containing phosphodiester bondsand are referred to as sense c-myc and antisense c-myc,respectively. P2s and its complementary antisense oligo werechimeric or phosphorothioate/phosphodiester bonds withphosphothioester bonds located between the first two and thelast two bases. These are referred to in the text as modifiedsense c-myc and modified antisense c-myc, respectively.These primers were used in embryo culture experiments.Immunohiochemistry. Embryos were recovered from the

reproductive tract in Whitten's medium. They were freed ofzona pellucida by a brief exposure to a 0.5% Pronase solutionin phosphate-buffered saline (PBS) and washed several timesin Whitten's medium (43). Embryos were placed onto poly(L-lysine)-coated slides by cytocentrifugation, air-dried for 30min, and fixed. For localization ofc-myc, embryos were fixedin cold methanol for 10 min or in 1% paraformaldehyde inPBS for 30 min. After hydration in PBS for 30 min, andblocking in 10%o normal goat serum, embryos were incubatedovernight at 40C in anti-c-myc antibody at a 1:500 dilution inPBS with 0.3% Triton X-100 (44). For localization of epider-mal growth factor receptor (EGF-R), embryos were fixed in10%6 neutral buffered formalin for 10 min and washed in PBS.They were then incubated in blocking solution (10%o normalgoat serum) for 10 min followed by incubation in rabbitpolyclonal antibody to mouse liver EGF-R (50 I&g/ml) (45) for24 hr at 4°C. Immunostaining was performed using a His-tostain-SP kit (Zymed Laboratories). The kit used a biotin-ylated secondary antibody, a horseradish peroxidase-streptavidin conjugate, and a substrate chromogen mixture.After immunohistochemistry, embryos were poststainedlightly with fast green for c-myc and hematoxylin for EGF-R.Red deposits indicated the sites of immunostaining. Controlexperiments consisted of incubation of embryos with normalrabbit serum or in the absence of the primary antibody.Embryo Culture. To study effects of antisense c-myc oligos

on preimplantation embryo development, two-cell embryoson day 2 (0800-0900 hr) of pregnancy were recovered fromseveral mice and pooled in Whitten's medium containing0.3% bovine serum albumin. Embryos were washed fourtimes and cultured in batches of 8-15 in microdrops (25 ,ul)of culture medium under silicon oil in an atmosphere of 5%C02/95% air at 37°C for 72 hr (43) in the presence or absenceof unmodified or modified antisense and sense c-myc oligosor with duplexes of the unmodified oligos. Oligos were addedat the beginning of the culture. To generate sense-antisenseduplexes, equal amounts of the sense (P2) and antisensec-myc oligos were mixed, heated to 800C for 5 min, andallowed to slowly cool to 370C for 5 hr prior to their additionto the embryo culture medium. Embryo development wasmonitored every 24 hr. At the end of the culture, the numberof embryos that developed to blastocysts was recorded andthe number of cells per blastocyst was determined (43). Incontrol cultures, >80% of the embryos developed into blas-

C=

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tocysts. Each experiment was repeated three or four timeswith a total of at least 40-50 embryos per experimental point.To determine c-myc protein and EGF-R in blastocysts

cultured in the presence of c-myc antisense or sense oligos,eight-cell (day 3) embryos were cultured (43) with either thec-myc sense oligo (P2) or antisense c-myc (7.5 ,uM) for 24 hr.At the end of the culture, those embryos that had formed ablastocoel were freed of zona pellucida, cytospun onto glassslides, and processed for immunolocalization of c-myc andEGF-R as described above.

RESULTSExpression of the c-myc gene in preimplantAtin mouse em-bryos was documented by detection ofc-myc IhRNA using theRT-PCR (Fig. 1) and by detection of c-'Yc protein usingimmunohistochemistry (Fig. 2). A search of the GenBank database for sequence identity with the c-myc primers used forRT-PCR (P1 and P2) indicated that no amplification of otherknown gene transcripts should occur under these reactionconditions. Amplification of a 398-bp product was predictedfrom the sequence of c-myc cDNA. The specific detection ofthis RT-PCR product by Southern blot hybridization using theinternal primer P3 as a probe further ensured the specificity ofthe assay. This primer (P3) has no significant sequence identitywith other members of the c-myc family. RT-PCR failed toamplify c-myc transcripts in RNA obtained from one-cellembryos (Fig. lb; day 1 of pregnancy). In contrast, low levelsof c-myc RT-PCR products were detected using two-cellembryo RNA (day 2), and high levels of RT-PCR amplifiedc-myc transcripts were detected using four-cell/eight-cell em-bryo RNA (day 3) and morula/blastocyst RNA preparations

bzx'.!

(day 4). These results were reproduced using two independentpreparations of RNA. No attempt was made to obtain quan-titative RT-PCR results. Immunohistochemical detection ofc-myc showed an absence of staining in one-cell and two-cellembryos, low-intensity nuclear staining in four-cell embryos,and intense nuclear staining at the eight-cell and morula/blastocyst stages (Fig. 2). Overall, these results suggest thatexpression of the c-myc gene may be initiated, albeit at lowlevels or only transiently, during activation of the zygoticgenome after the first cleavage (two-cell). In contrast, c-mycexpression is heightened and apparently constitutive after thethird cleavage (eight-cell) and remains high during differenti-ation of morulae into blastocysts.That c-myc plays an important role in preimplantation

development was suggested by the finding of severely atten-uated blastocyst formation from two-cell embryos cultured inthe presence of a low concentration (7.5 AuM) of the antisensec-myc oligo (Fig. 3). This antisense c-myc oligo was comple-mentary to the region of c-myc mRNA beginning with thetranslation initiation codon and spanning the next seven aminoacids (reverse complement of P2 in Fig. la). When included atcomparable concentrations (7.5 A&M) in the culture medium,the sense c-mnyc oligo (P2) and the duplex of these. antisenseand sense oligos had little effect on blastocyst formation.However, embryos cultured *ith only 4-fold higher concen-trations of any of the single or double-strand unmodifiedc-myc oligos arrested dev#lppment before the blastocyststage. These embryos arrikted at the two-cell or four-cellstage, whereas embryos cult!Ired With low levels of antisensec-myc arrested developme the eight-cell/morula stage anddid not proceed to the blastocyst stage. This suggested thepossibility that a nonspecific toxic effect ofDNA in the culture

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FIG. 2. Immunohistochemical localization of c-myc protein in preimplantation mouse embryos on days 1-4 of pregnancy. Embryos werecytospun onto poly(L-lysine)-coated slides and fixed in 1% paraformaldehyde in PBS. After hydration in PBS and blocking in 109% normal goatserum, embryos were incubated in anti-c-myc antibody (44). Blastocysts were counterstained with fast green. Blastocysts incubated innonimmune rabbit serum did not show specific staining (data not shown). Red (dark) deposits in the nucleus indicate the site of immunoreactivec-myc. Embryonic developmental stages shown are one-cell (a), two-cell (b), four-cell (c), eight-cell (d), decompacted eight-cell (e), andblastocyst (f). (x400.) Decompaction was achieved by incubating embryos in calcium-free medium. ICM, inner cell mass; Tr, trophectoderm.

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40

20

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CONCENTRATION OF OLIGO

FIG. 3. Development of preimplantation embryos cultured with c-myc oligos. Two-cell embryos were cultured in batches of 8-15 in 25-tilmicrodrops of culture medium under silicon oil at 370C for 72 hr in the presence or absence of the indicated concentrations of sense, antisense,or sense-antisense duplex c-myc oligos (see Fig. la). In other experiments, the culture medium contained the indicated concentrations ofmodified antisense or sense c-myc oligos. These modified oligos had phosphothioesters located between the first two bases and the last two basesof the oligo. Oligos were added at the beginning of the culture. In control cultures, >80% of the embryos developed into blastocysts. Eachexperiment was repeated three or four times with a total of 40-50 embryos per experimental point.

medium could influence the results. However, it was alsonoted that in vitro development offreshly recovered eight-cellembryos to the blastocyst stage was specifically inhibited (36%blastocyst formation) when cultured in medium containing 7.5,uM antisense c-myc, whereas the same concentration of sensec-myc (P2) had little effect (78% blastocyst formation).

Examination ofblastocysts that developed in the presence ofantisense c-myc showed the complete absence ofc-myc proteinin the cell nuclei, whereas blastocysts that developed in mediumcontaining sense c-myc stained positively for the protein (Fig.4a). The effects of antisense c-myc appeared to be specific,since the abundance of immunoreactive EGF-Rs in similarlytreated blastocysts remained unaltered (Fig. 4b). Those blas-tocysts that developed during culture with the antisense c-mycwere not examined for the potential to continue development.These results are consistent with the suggestion that antisensec-myc acts to specifically attenuate synthesis of this protein.

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To further validate the antisense c-myc effects, experi-ments were carried out using chimeric phosphorothioate/phosphodiester oligos. The inclusion ofphosphothioesters inoligos has been reported to increase their stability and uptake(26). As shown in Fig. 3, essentially all embryos (98%)cultured in the presence of a low concentration (7.5 AM) ofthe modified antisense c-myc oligo failed to develop to theblastocyst stage from the two-cell stage. In contrast, thoseembryos cultured in 4-fold higher concentrations (30 AM) ofthe modified sense c-myc oligo developed to the blastocyststage with a frequency (80-90%o) indistinguishable from thatof embryos cultured in the absence of oligo.

DISCUSSIONThese data provide evidence that activation of the zygoticc-myc gene and accumulation of this nuclear protein coin-cides with a critical function ofthis protein particularly at the

FIG. 4. c-myc protein and EGF-R in blastocysts cultured in the presence ofc-myc antisense or sense oligos. Eight-cell embryos were culturedwith either the unmodified c-myc sense oligo (P2) or antisense c-myc (7.5 ,uM) for 24 hr, and at the end of the culture blastocysts that haddeveloped were cytospun onto poly(L-lysine)-coated slides. (a) Embryos were fixed in 1% paraformaldehyde in PBS and incubated in anti-c-mycantibody (44). Photomicrographs of c-myc immunostaining in representative blastocysts that had developed in the presence of sense (Left) orantisense c-myc (Right) are shown at x300. (b) Embryos were fixed in 10%1 neutral buffered formalin in PBS and incubated in anti-EGF-Rantibody (50 Ag/ml) (45). Photomicrographs ofEGF-R immunostaining in representative blastocysts that had developed in the presence of sense(Left) or antisense c-myc (Right) are shown at x300.

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eight-cell/morula stage of development of preimplantationmouse embryos. Thus, c-myc is apparently involved in thedifferentiation and/or late cleavages of embryos. Althoughthe data suggest that this protein may not be critical for thefirst few cleavages of the fertilized egg, it is conceivable thatuptake and/or stability of the oligo may differ between theearly cleavage stage embryos as well as the eight-cell/morulaand blastocyst. A lack of detection ofeither the c-myc proteinor mRNA in these early embryos could be attributed to thetransient expression of this gene during each of the first twocleavages. However, this seems unlikely since the majority ofcells stained positively for c-myc at later stages ofembryonicdevelopment (morulae and blastocysts). Further investiga-tion will be required to resolve this matter. The mechanismby which c-myc expression in the preimplantation embryo isregulated is not known. It is possible that growth factors suchas EGF/transforming growth factor a of reproductive tract(42) or embryonic origin (40) may play a role. EGF has beenshown to up-regulate c-myc expression in cultured cells (46).During embryonic development ofthe mouse, EGF receptorsare first detected at the eight-cell stage (43), and this growthfactor has beneficial effects on growth and differentiation ofmouse embryos into blastocysts in vitro (43). However, thepossibility that other growth factors may be involved shouldnot be ignored.These studies establish that antisense oligos can be em-

ployed as an effective approach to attenuating gene functionduring early mammalian embryogenesis. Although directmicroinjection of antisense RNAs or DNAs as a means ofblocking specific gene expression in mammalian and non-mammalian oocytes and early embryos has met with successand failure (47-52), a recent attempt to alter P-glucuronidasegene expression in the preimplantation mouse embryo by theaddition ofantisense oligos to the culture medium failed (53).Therefore, the approach reported here may not be gerierallyapplicable. The success of this approach may be largelydependent on the gene being examined, and we hypothesizethat the extremely short half-life ofc-myc mRNA and protein(20-22) may account in a large part for the success of theseexperiments. However, the studies herein also suggest thatthe concentration and the structure of the antisense oligoinfluence its effects on embryogenesis.

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