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《Molecular Cell》2010年5月14 (6)

《Molecular Cell》2010年5月14 (6)
《Molecular Cell》2010年5月14 (6)

2073

2074WRENZYCKI ET AL.

K-ATPase?1subunit(Na/K),E-cadherin(E-cad),zonula occludens protein-1(ZO-1),desmocollin II(Dc II),plako-philin(Plako);trophoblastic function,i.e.,interferon?(IF?);and glucose transport,i.e.,glucose transporter-1,-3, -4(Glut-1,-3,-4)were analyzed in intact in vitro-produced blastocysts from Days7and8and isolated ICM derived thereof.

MATERIALS AND METHODS

In Vitro Production of Bovine Embryos

Bovine embryos were produced as described recently[16].Brie?y, ovaries from a local slaughterhouse were transported to the laboratory in Dulbecco PBS(D6650,Sigma Chemical Co.,St.Louis,MO)at25?C–30?C.Cumulus-oocyte complexes(COCs)were isolated via slicing[20]. Category I COC(with a homogenous evenly granulated cytoplasm pos-sessing at least three layers of compact cumulus cells)and category II COC(with less than three layers of cumulus cells or partially denuded but also with a homogenous evenly granulated cytoplasm;[21])were pooled in TCM air(TCM199containing L-glutamine and25mM Hepes[Sigma] supplemented with22?g/ml pyruvate,350?g/ml NaHCO3,50?g/ml gentamicin,and0.1%bovine serum albumin[BSA,fraction V,A9647, Sigma]).

For maturation in vitro,TCM199containing L-glutamine and25mM Hepes served as the basic medium.One milliliter was supplemented with 22?g pyruvate,2.2?g NaHCO3,and50?g gentamicin.For oocyte mat-uration,this medium was supplemented with10IU eCG and5IU hCG (Suigonan;Intervet,To¨nisvorst,Germany)and10%oestrus cow serum (collected at Day1of standing oestrus).COC were divided in groups of 20–25,transferred into100?l maturation drops under silicone oil,and cultivated in a humidi?ed atmosphere composed of5%CO2in air at39?C for24h.

Following in vitro maturation,COC were rinsed in fertilization medi-um(Fert-TALP supplemented with6mg/ml BSA)and fertilized in Fert-TALP containing10?M hypotaurine(Sigma),1?M epinephrine(Sigma), 0.1IU/ml heparin(Serva,Heidelberg,Germany),and6mg/ml BSA.Fro-zen semen from one bull with proven fertility in in vitro fertilization(IVF) was used.For IVF,semen was prepared by the modi?ed‘‘swim-up’’pro-cedure[22,23].Brie?y,semen was thawed in a waterbath at37?C for1 min.After swim-up separation in Sperm-TALP containing6mg/ml BSA for1h,the semen was washed twice by centrifugation at350g and36?C for10min before being resuspended in Fert-TALP supplemented with heparin and BSA.The?nal sperm concentration added per fertilization drop was1?106sperm/ml.Fertilization was initiated during a19h coincubation under the same temperature and gas conditions as described for maturation.

Presumptive zygotes were cultured in30?l of synthetic oviduct?uid (SOF)supplemented with10%estrous cow serum after complete removal of the adhering cumulus cells by repeated pipetting.Embryos were cul-tured in vitro in a mixture of7%O2,88%N2,and5%CO2(Air Products, Hattingen,Germany)in Modular incubator chambers(615300,ICN Bio-medicals,Inc.,Aurora,OH).Expanded blastocysts were harvested at either Day7or Day8of development(day0?IVF)as intact embryos or for isolating the ICM.Only blastocysts of morphological grade I[24]were included in this study.After washing three times in PBS containing0.1% polyvinyl alcohol(PVA),all intact embryos or isolated ICM were stored individually at?80?C in a minimum volume(5?l or less)of medium until RNA extraction.

Differential Staining of ICM and TE Cells

and ICM Isolation

For determination of total cell numbers and the ratio of ICM and TE cells in in vitro-produced bovine embryos,the differential staining pro-cedure was performed with a representative sample of Day7and Day8 blastocysts according to the protocol described by Eckert and Niemann [25]with minor modi?cations.Brie?y,after removal of the zona pellucida with1.0%pronase(Type XXV,Sigma,P6911)in PBS(w/v),the embryos were incubated in trinitrobenzene sulfonic acid(Sigma,P883)for10min on ice,then washed in TCM air and incubated in antidinitrophenyl BSA (anti-DNP BSA;61-006,ICN Biochemicals,Eschwege,Germany)diluted 30:70in PBS at37?C for30min.After washing in PBS plus PVA,com-plement lysis was induced by incubating the embryos in guinea pig com-plement(Sigma,S1639)diluted1:4in PBS plus PVA supplemented with propidium iodide(Sigma,P4170,10?g/ml)at37?C for20min,followed by brief washing and?xation in ice-cold ethanol.The inner nuclei were stained with Hoechst33342(Bisbenzimid,Sigma,B2261,10?g/ml in ethanol)for10min.Stained embryos were mounted into100%glycerol. Under a?uorescence microscope(excitation?lter at420nm,barrier?lter at365nm),the outer TE cells were identi?ed by the pink?uorescence of propidium iodide,whereas the ICM cells were recognized by their blue ?uorescence of the bisbenzimide.Embryos in which clear evaluation failed or that were disrupted during the staining procedure were discarded.

ICMs were isolated employing the same protocol as described above. After removal of the zona pellucida,incubation in trinitrobenzene sulfonic acid and anti-DNP BSA,lysis of the outer cells was induced via guinea pig complement diluted1:4,but without propidium iodide.To ensure com-plete removal of adhering TE cells from the ICMs,only embryos in which all TE cells were evenly lysed were processed.The ICMs were liberated from lysed TE cells by gentle passage through a?ne hand-drawn pipette. Finally,the isolated ICMs were washed three times in PBS supplemented with0.1%PVA before being frozen at?80?C.Intact blastocysts from the same IVP run were sham-treated in parallel by removing the zona pellu-cida.

Determination of the Relative Abundance

of Developmentally Important Gene Transcripts

in Bovine Embryos

Poly(A)?RNA was isolated from single blastocysts or single isolated ICMs as described recently[26]and was used immediately for reverse transcription(RT),which was carried out in a total volume of20?l using 2.5?M random hexamers(Perkin-Elmer,Vaterstetten,Germany).Prior to RNA isolation,1pg of rabbit globin RNA(BRL,Gaithersburg,MD)was added as an internal standard and was analyzed simultaneously.The rabbit globin mRNA represents a mixture of the?-and?-chains derived from polyribosomes of reticulocytes and was puri?ed via oligo-dT cellulose chromatography.No products were obtained when the transcripts for the gene of choice were analyzed.Globin with and without preparation(globin ?prep)served as recovery control.Previously,the validity of this ap-proach had been proven and revealed ef?cient ampli?cation of the globin standard and the RNA of choice[27].The reaction mixture consisted of 1?RT buffer(50mM KCl,10mM Tris-HCl,pH8.3,Perkin-Elmer);5 mM MgCl2;1mM of each dNTP(Amersham,Brunswick,Germany);20 IU RNase inhibitor(Perkin-Elmer);and50IU MuLV reverse transcriptase (RT)(Perkin-Elmer).The mixture was overlaid with mineral oil to prevent evaporation.The RT reaction was carried out at25?C for10min,42?C for1h,followed by a denaturation step at99?C for5min and?ash cooling on ice.Polymerase chain reaction(PCR)was performed with embryo equivalents(percentage of the volume from the RT reaction employing one embryo in a de?ned volume)as described in Table1from different embryos or ICMs generated in different IVP runs in a?nal volume of50?l of1?PCR buffer(20mM Tris-HCl,pH8.4,50mM KCl,Gibco BRL, Eggenstein,Germany);1.5mM MgCl2;200?M of each dNTP;and1?M of each sequence-speci?c primer(globin,0.5?M)using a PTC-200 thermocycler(MJ Research,Watertown,MA).To ensure speci?c ampli-?cation,a‘‘hot start’’PCR was employed by adding1IU Taq DNA polymerase(Gibco)at72?C.PCR primers were designed from the coding regions of each gene sequence using the oligo program.The sequences of the primers used,the annealing temperatures,the fragment sizes,and the sequence references have been summarized in Table1.

The PCR program employed an initial step of97?C for2min and72?C for2min(hot start)followed by different cycle numbers(see Table1)of 15sec each at95?C for DNA denaturation,15sec at different temperatures for annealing of primers,and15sec at72?C for primer extension.Due to the very low amount of starting material(?5ng RNA),35–39PCR cycles are required for an ef?cient ampli?cation of embryonic transcripts[27]. The last cycle was followed by a5min extension at72?C and cooling to 4?C.As negative controls,tubes were prepared in which RNA or reverse transcriptase was omitted during the RT reaction(data not shown).

The RT-PCR products were subjected to electrophoresis on a2%aga-rose gel in1?TBE buffer(90mM Tris,90mM borate,2mM EDTA, pH8.3)containing0.2?g/ml EtBr.Further EtBr in the same concentration was added to the running buffer.The image of each gel was recorded using a CCD camera(Quantix,Photometrics,Mu¨nchen,Germany)and the IP Lab Spectrum program(Signal Analytics Corporation,Vienna,VA). The intensity of each band was assessed by densitometry using an image analysis program(IP Lab Gel;Scananalytics,Fairfax,VA).The relative amount of the mRNA of interest was calculated by dividing the intensity of the band for each developmental stage by the intensity of the globin band for the corresponding stage.Experiments were repeated with at least 10embryos or ICMs for each mRNA.The RA in the ICM isolated from

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SPATIAL GENE EXPRESSION IN BOVINE EMBRYOS

TABLE1.Primers used for PCR.

Genes Primer sequences and positions Annealing temperature

(?C),cycle number,and

embryo equivalent

Fragment size

(bp)

Sequence references

(EMBL accession no.)

Globin5?primer(241–260)?

GCAGCCACGGTGGCGAGTAT

3?primer(555–657)?

GTGGGACAGGAGCTTGAAAT 60?27

0.05

25766

(X04751)

Na/K-ATPase(Na/K)5?primer(2882–2903)?

ACCTGTTGGGCATCCGAGAGAC

3?primer(3196–3217)?

AGGGGAAGGCACAGAACCACCA 58?31

0.1

33667

(NM?012504)

E-cadherin(E-cad)5?primer(1486–1515)?

CTCAAGCTCGCGGATAACCAGAACAAAGAC

3?primer(1785–1841)?

AGGCCCCTGTGCAGCTGGCTCAAATCAAAG 55?32

0.2

33268

(X06339)

Desmocollin II(Dc II)5?primer(2085–2109)?

CTCCTGGCGATGACAAAGTGTATTC

3?primer(2503–2527)?

GCCGATCCTCTTCCTTCGTAGTTAT 57?31

0.2

443/39769

(M81190)

Plakophilin(Plako)5?primer(1337–1361)?

CCCGTGGACCCCGAGGTCTTCTTCA

3?primer(1580–1604)?

CGGTGTAGGCGTTGCGGGCGTTGTA 64?35

0.4

26870

(Z37975)

Zonula occludens—protein 1(ZO-1)5?primer(5922–5921)?

CACAGTTTGGCACAGCCTCCTGAGTTTGAC

3?primer(6350–6379)?

CCGGGAAGACACTTGTTTTGCCAGGTTTTA

61?34

0.2

522/478/45871

(L14837)

Interferon tau(IF?)5?primer(420–443)?

GCTATCTCTGTGCTCCATGAGATG

3?primer(755–778)?

AGTGAGTTCAGATCTCCACCCATC 57?35

0.5

35972

(G163764)

Glucosetrans porter-1 (Glut-1)5?primer(1609–1638)?

AGGAGCTGTTCCACCCCCTGGGAGCTGACT

3?primer(1906–1935)?

TGTGGGTGAAGGAGACTCTGGCTGATAAAA

59?32

0.1

32773

(M60448)

Glucose transporter-3 (Glut-3)5?primer(1129–1152)?

CCTTGGAGGGATGGCTTTTTGTTC

3?primer(1364–1387)?

CGTGGCTGAGGGGAAGAGCAGTCC

58?35

0.1

25974

(L39214)

Glucose transporter-4 (Glut-4)5?primer(1663–1692)?

GGAGCAGGAAGTGAAACCCAGCACAGAACT

3?primer(1919–1939)?

GCTAACCACAACACAAATAATCCAAGAGGT

59?39

0.5

27775

(D63150)

blastocysts from that of contemporary intact blastocysts(RA TE?RA intact blastocyst RA ICM)in the same replicate IVP run.RA was calcu-lated on a per cell basis for intact embryos or isolated ICMs,respectively, taking into account the average cell number found for blastocysts from Days7and8.Blastocysts from the same IVP run were used either as intact embryos or after ICM isolation.The calculated repeatability(0.90) and the average accuracy(0.70)of the assay allowed calculation of statis-tically signi?cant differences between treatment groups for each transcript from a minimum of eight replicates[28].

For each pair of gene-speci?c primers,semilog plots of the fragment intensity as a function of cycle number were used to determine the range of cycle number over which linear ampli?cation occurred,and the number of PCR cycles was kept within this range[26].Since the total ef?ciency of ampli?cation for each set of primers during each cycle is not known, such assays can only be used to compare RAs of one mRNA among different samples[29].This semiquantitative RT-PCR assay yields sensi-tive and highly reproducible results from pooled and single embryos and compares favorably with real-time PCR[30].

Statistical Analyses

Cell numbers and RAs were analyzed using the SigmaStat2.0(Jandel Scienti?c,San Rafael,CA)software package.After testing for normality (Kolmogorov-Smirnov test with Lilliefor correction)and testing for equal variance(Levene median test),an analysis of variance(ANOVA)followed by multiple pairwise comparisons using the Tukey test was employed. Differences of P?0.05were considered to be signi?cant.RESULTS

Allocation of ICM Versus TE Cells in Expanded IVP Blastocysts at Day7or Day8of Development

A total of30Day7and19Day8expanded IVP blas-tocysts were differentially stained and possessed an average of147?6and146?8cells,of which58?4and64?5were allocated to the ICM,corresponding to an ICM/total cells ratio of39?2%and43?2%,respectively.No signi?cant difference was detected between the two differ-ent groups.These average cell numbers were used when calculating the RA for intact embryos and ICMs on a per cell basis.

Spatial Expression of Transcripts

Representative gel photographs of a semiquantitative RT-PCR assay of the analyzed gene transcripts in single expanded bovine blastocysts from Day7or Day8of de-velopment and ICMs isolated thereof,as well as the cor-responding globin bands,are shown in Figure1.

As shown in Figure2,the examined transcripts showed different patterns of distribution within the two cell line-ages.Plako and IF?transcripts were not detected in isolated

2076WRENZYCKI ET AL.

FIG.1.Representative gel photographs of a semiquantitative RT-PCR analysis of various developmentally important gene transcripts in intact in vitro-produced bovine blastocysts([A1],[A3],[B1],[C1])or isolated ICMs([A2],[A4],[B2],[C2]).Blastocyst expansion occurred in these embryos at either Day7([A1],[A2],[B1],[B2])or Day8([A3],[A4],[C1],[C2]).Globin?prep(globin?prep?globin present during RNA extraction procedure; globin?prep?globin RT-PCR without RNA extraction procedure)indicates the control of RNA recovery.

2077

SPATIAL GENE EXPRESSION IN BOVINE

EMBRYOS FIG.2.Distribution of the various transcripts within the two cell lineages (TE ?dark gray,ICM ?light gray)of Day 7or Day 8

blastocysts.

FIG.3.Effects of timing of blastocyst ex-pansion on relative mRNA abundances (means ?SEM)in intact expanded bovine blastocysts from Day 7(open bars)or Day 8(black bars)as well as in ICMs isolated from Day 7(light gray bars)or Day 8(dark gray bars)blastocysts.The relative abundance of each mRNA in the TE of Day 7(horizontal stripe)or Day 8(vertical stripe)blastocysts was calculated by sub-tracting the amount of the ICM from that of the intact embryo.Bars with different superscripts within each gene transcript differ signi?cantly ([A]:[B]ICM vs.TE in Day 7blastocysts;[a]:[b]ICM vs.TE in Day 8blastocysts;P ?0.05).Signi?cant differences (P ?0.05)in the TE of blasto-cysts expanding at either Day 7or Day 8are indicated by an asterisk (*).The rela-tive abundances have been calculated on a per cell basis for intact embryos and iso-lated ICMs as well.

ICMs,indicating that these mRNAs are restricted to the TE.Similar expression patterns within the ICM and TE were detected for Na/K,E-cad,ZO-1,and Glut-3mRNA,where-as Dc II,Glut-1,and Glut-4mRNA showed a higher (P ?0.05)level of expression within the TE than in the ICM.Effect of the Age of the Blastocyst During Expansion on the Relative Abundance of Developmentally Important Gene Transcripts

Alterations in mRNA abundances of the speci?c gene transcripts in relation to the occurrence of blastocyst ex-pansion,either at Day 7or Day 8,are depicted in Figure 3.Signi?cant differences between TE cells from blastocysts that showed expansion at either Day 7or Day 8were de-termined for the RA of Na/K,E-cad,Dc II,Plako,and ZO-

1transcripts.The RA of Dc II,Glut-1,and Glut-4were signi?cantly decreased in the ICM compared with the TE at Day 7.Similarly,the RA of Na/K,Dc II,Glut-1,and Glut-4at Day 8of development was signi?cantly decreased in the ICM versus TE.Interestingly,no differences were observed in ICMs originating from blastocysts expanded at either Day 7or Day 8.DISCUSSION

The present study is the ?rst to investigate the effects of timing of blastocyst expansion on the spatial expression pattern of genes speci?cally involved in compaction/cavi-tation,trophoblastic function,and glucose transport.No dif-ferences were observed in the cell number of blastocysts with an expanded blastocoele either at Day 7or Day 8,indicating differences in the speed of developmental pro-gress.Previously,Day 7blastocysts had a signi?cantly higher cell number than those appearing at Day 8[2,31,32].We have shown that bovine IVP-expanded blastocysts from Day 7are superior in their in vivo survival after freez-ing and thawing than their Day 8counterparts [10].The cell numbers found in the present study for IVP embryos compared favorably with those determined for in vivo-de-rived expanded blastocysts [33],indicating that expanded blastocysts of excellent quality had been analyzed in the present study and no major differences between Day 7and Day 8embryos could be found.However,sex-related dif-ferences cannot be ruled out.

Accumulation and composition of ?uid in the blasto-

2078WRENZYCKI ET AL.

coele during early murine embryonic development is crit-ically regulated by Na/K-ATPase activity and is essential for differentiation of ICM and TE cell types[34].In bovine embryos produced in vitro,transcripts encoding multiple isoforms of Na/K-ATPase are expressed throughout early development[35].In the present study,Na/K transcripts were detected in the ICM and TE from expanded blasto-cysts,which is consistent with the?nding of?1-subunit polypeptide in TE and ICM cells of bovine blastocysts[36].

E-cad-mediated cell to cell adhesion is associated with compaction[11,37].E-cad transcripts and protein were found in the ICM and TE of expanded bovine blastocysts [38,39].In the present study,mRNA for ZO-1was found in both the TE and ICM cells of the bovine blastocyst.ZO-1protein was detected as a continuous ring at the apical points of TE cell contact in bovine and murine embryos [39,40].ZO-1mRNA within ICM cells may be inherited from the parent cell undergoing differentiation into TE or stem from de novo transcription.

In contrast to ZO-1,in the present study desmocollin II (Dc II)was derived predominantly from the TE cells of the bovine blastocyst.A similar expression pattern has been detected in murine embryos[41].Desmocollin II is in-volved in the formation of desmosomal junctions that play a critical role in stabilizing the TE during blastocyst ex-pansion[41,42].Plako1is a major component of the des-mosomal plaque from strati?ed and epithelia[43]and is involved in desmosome organization and stability as well as in the regulation of desmosome assembly[44].In ex-panded bovine blastocysts,Plako mRNA is restricted to the TE.Protein data are not yet available.

Bovine embryos begin to express IF?as soon as the blastocyst forms[45].IF?production is primarily depen-dent on the presence of a functional TE[46].These?ndings are consistent with the present results as no differences were detected in the RA of IF?mRNA in expanded blas-tocysts produced either at Day7or Day8.However,a negative relationship between early IF?production and de-velopmental competence has been reported[47].Compar-ing in vivo-and in vitro-derived blastocysts,it was sug-gested that an early and high amount of IF?mRNA indi-cates poor quality of the bovine embryo[16,48].

Starting with the blastocyst stage,bovine embryos be-come dependent on aerobic metabolism and acquire the ca-pacity to utilize glucose[17].These changes in the role of glucose during preimplantation development indicate a close relationship between glucose metabolism and the ex-pression of glucose transporters[48].In the present study, we have shown that bovine Glut-1and Glut-4transcripts are expressed at higher levels in TE cells compared with ICM cells,whereas a similar expression pattern was found for Glut-3transcripts in the two lineages.Glut-3and Glut-4were selected for the present study because recent data indicated that both transcripts are susceptible to environ-mental changes,suggesting a critical role in the formation and maintenance of the bovine blastocyst[19].In the mouse,the well-orchestrated spatial expression pattern of glucose transporter mRNAs plays a critical role throughout preimplantation development[49].Glut-1protein was de-tected in the ICM and TE of murine blastocysts,whereas Glut-3protein was exclusively found in TE cells,and Glut-4was never determined in early murine and human embryo development[50–53].Glut-8expression and translocation are correlated with blastocyst survival in the mouse[54]. These data indicate that spatial glucose transporter expres-sion is regulated in a species-speci?c manner.Murine and bovine embryos have a different temporal expression pat-tern with regard to glucose transporters[18,52].But it has to be taken into account that protein data are not yet avail-able for preimplantation bovine embryos.Recently,we have shown that glucose transporter mRNA expression is affected by culture conditions with a decrease of Glut-1and an increase in Glut-3compared with in vivo embryos[16, 19,28].A persistent decrease in glucose transport may re-sult in enhanced apoptosis that is potentially associated with fetal malformation or miscarriage[55,56].Experi-ments blocking Glut-3expression via antisense oligodeox-ynucleotides have shown that Glut-3expression might have physiological functions additional to its activity in glucose transport in murine blastocyst formation[51].

The genes involved in compaction and cavitation ana-lyzed in the present study show a signi?cantly higher tran-scription in TE cells isolated from Day8-expanded blas-tocysts compared with TE cells from Day7embryos,in-dicating that expression of these genes is correlated with the timing of blastocyst expansion.Increased transcription is indicative for stress response and poor quality of the early embryo[26].The present results support this observation. Previously,it was shown that in vitro-produced expanded blastocysts appearing at Day7have a better cryosurvival than their in vivo-and in vitro-derived Day8counterparts [4,10],which is likely attributed to a favorable ultrastruc-ture of the TE[57–59].These?ndings suggest a different inherent quality of expanded blastocysts from Day7versus those from Day8.

A well-controlled expression of genes is essential for the regular formation of the two lineages of the blastocyst.Al-terations in the expression pattern of leukemia inhibitory factor(LIF)and its receptor subunits in in vitro-produced bovine blastocysts have been detected and were associated with a perturbated status of early differentiation[25].Dur-ing blastocoele formation the LIF-LIF receptor system is downregulated probably to slow down proliferation and to allow enough time for the organization of cell differentia-tion[2,32].Perturbations in cell allocation in the blastocyst may lead to either early embryonic death or the formation of fetal anomalies associated with early abortions[60].In cloned bovine blastocysts an aberrant allocation of ICM and TE cells has been determined[61].A higher incidence of placental anomalies is observed in both early and late gestation and in pregnancies derived from cloned or in vi-tro-produced embryos[62],which may have originated from early perturbations in blastocyst formation.Similarly, expression of genes that were exclusively found in the TE was affected by in vitro culture and nuclear transfer[16, 28].Recently,an unequal methylation pattern was found between ICM and TE regions,suggesting a widespread gene dysregulation in extra embryonic regions,thereby re-sulting in placental dysfunction[63].Collectively,the above?ndings indicate that deviations from the normal spa-tial expression pattern early in development may have pro-found effects for the outcome of pregnancy.

In conclusion,the present study has identi?ed genes that are exclusively expressed in the ICM or TE.Their expres-sion was affected by timing of blastocyst expansion.Future studies employing in vivo IVP embryos from different cul-ture systems or nuclear transfer-derived embryos will aid in elucidating whether in vitro culture or cloning alter the spatial expression pattern of genes in both cell lineages nec-essary for normal development.The need for further studies also arises from the numerous unsuccessful attempts to gen-erate and cultivate true embryonic stem cells from the

2079 SPATIAL GENE EXPRESSION IN BOVINE EMBRYOS

ICMs of preimplantation bovine embryos[64,65].The ?ndings of the present study emphasize the critical role of an adequate spatial gene expression in preimplantation bo-vine embryo development.

ACKNOWLEDGMENTS

The authors are grateful to K.Korsawe and S.Wilkening for their skilled technical assistance.

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