Sex diagnosis of ovine and bovine embryos by enzymatic amplification and digestion of DNA from the ZFY/ZFX locus
Peter Bredbacka and JaanaPeippo
Bredbacka, P.& Peippo,J. 1992.Sex diagnosisof ovine and bovine embryos by enzymatic amplificationanddigestionofDNAfrom theZFY/ZFXlocus.Agric.
Sei.Finl. 1: 233-238. (Agric.Res. Centre ofFinland., Dept.Anim, Breed.,SF-31600 Jokioinen,Finland.)
APCR-based sex determination assay forsheepand cattle embryoswasdeveloped usingmouseembryosforoptimizing theprotocol. Sampleswerelysedeither enzyma- ticallyorby alkaline treatment followedby enzymatic amplificationofDNAfrom the
ZFY/ZFXlocus. Sexdiagnosiscould be done after thedigestionof theamplifiedpro- duct byrestriction endonucleases. Ovine and bovine embryos could be sexed from biopsies assmallas1-4cells. Someembryosweresplitinto2-4 sections,whichwere amplified separately.Blind trials with such samples demonstrated that the methodwas highlyaccurate,evenwhenembryo biopsy wasdone under farm conditions. The pro- tocol involves anin-built control. This eliminates the need for autosomal controlpri- mers, which often inhibit theamplificationof theY-chromosome-specific DNA,es- peciallywhenasmall amount of template is used.
Keywords:sexing, embryo, sheep, cattle, polymerase chainreaction,restrictionfrag- ment length polymorphism
Introduction
The production of offspring of the desiredsex from livestock species is becoming areality. Although the method of choice would be the separation of X- and Y-chromosome-bearing sperm priortoAI, the only application thatcanbe used in practical condi- tions at present is to sex embryos in connection with embryo transfer. Various approaches have been usedto determine thesex of embryos (for a review, see Van Vlietetal. 1989), and atpresent the technique utilizing the polymerase chain reaction(PCR)seemstobe themosteffectiveone.
PCR involves the enzymatic amplification of DNA with the help of oligonucleotide primers (Saikietal. 1985).By annealingtospecific sites of the genomicDNA,the primers define the initiation site of DNA synthesis. When primers anneal on
opposite DNA strands, the intervening DNA sequencecan be amplified in anexponential man- ner. This is possible by repeating each amplifica- tion cycle, which involves heat denaturation of the DNA,primer annealing and DNA synthesis.
The emergence of thermostabile polymerases for DNA synthesis (Saiki etal. 1988) has greatly in- creased the applicability ofPCR. Thegreaterspeed has made it possible to sex embryos on the same day as embryo recovery and transfer are carried out. Several authors have reported successfulam- plification of male-specific DNA from embryo biopsies (Handyside etal. 1989,Bradburyetal.
1990, Herretal. 1990a,b,Schröderetal. 1990, Bredbacka etal. 1991, Peuraetal. 1991, Setia-
budiand Gustavsson 1991), but further develop- ment is needed to make embryo sexing a more straightforward anda morereproducible technique.
Agric. Sei.Finl. 2(1992)
The lack of male-specific amplification product can be caused either by afemale biopsy servingas the target orby unsuccessful amplification condi- tions. To test the amplification success, an autosomal primer paircan be addedtothe reaction mix. Flowever, this usually leads to a decreased yield of the male-specific product, sometimes at levels below distinction. Usually this isnotaprob- lem when a large amount of DNA is availableas template, but for sexing embryos onlyafew cells of the embryo canbe sacrificed.Thus, the testing of the method should be done witha limited number ofcells,preferably with embryonic cells. This in- troduces another problem, the availability of emb- ryonic material for optimizing PCR conditions.
Livestock embryos are expensive, and embryos from other speciescanusuallynotbe used because the male-specific primers tendtobe more orless species-specific.
This paper presents a PCR protocol for sexing bovine and ovine embryos without the need for autosomal control primers. The amplified sequence is within the ZFY locus. This locus ispresent onthe Y chromosome of all placental species and has a homologue on the X chromosome (Page et al.
1987). In themouse, there aretwo homologues on theY-chromosome, one onthe X-chromosome and a fourth autosomal homologue. Male and female productscan be identified duetodifferences in the restriction fragment length polymorphism (RFLP) of the ZFY/ZFX locus (Aasen and Medrano 1990). Mouse embryos were used for optimizing and testing the protocol.
Material and methods
The oligonucleotide primers were modified after Aasen and Medrano (1990) to match themouse ZFY sequence as reported by Ashworth et al.
(1989). The primer sequenceswerethe following:
Pl-SEZ: s'-ATA ATC ACA TGG AGA GCC ACA AGC T- 3'
P3-3MZ: s'-GAG CCT CTT TGG TAT CTG AGA AAG T- 3'
Initial optimization of PCR parameters was done
using DNA extracted from tails of female and male mice after overnight incubation in proteinase K fol- lowed by phenol/chlorophorm and ethanol precipitation. The sample amount was approxi- mately 80 pg.
Further optimization was carried out with embryos recovered from mice superovulated by standard procedures. Fresh, refrigerator-stored or frozen cattle and sheep embryos were biopsied using the 'scratched-bottom' technique (Bred-
backa 1991). Biopsies were transferred tomicro- tubes witha micropipette havinganinner diameter only slightly larger than the diameter ofanembryo.
In some instances, siliconized pipettes were used.
Up tofour portions from each embryowere separ- ately amplified and the accuracy of the methodwas measured inablindtest.
Prior to PCR, the cells were lysed either by 1- hour incubation withproteinase Kat37°C followed by inactivationat99°C (8 minutes),oralternatively by a modification of the sperm lysis described by Cui etal (1989). In the lattermethod, the sample, which is in 1 pi of medium (PBS+ 4 mg/ml polyvi- nylpyrrolidone), is added to 2.5 pi of 200 mM K.OH/50 mM dithiotreitol and incubated for 10 minutes at 65°C, followed by neutralization with 2.5 pi of 900mM Tris-HCI (pH 8.3)/200 mM HCI.
The final PCR mixture contained the sample in 10-100 mM Tris-HCI (pH 8.8), 1.5 mM MgCl2, 50 mMKCI, 0.01% gelatin, 0.1% Triton X-100, 0.2 mM dNTPs (dATP, dTTP, dGTP, dCTP), 1.25 1U Taq polymerase (Promega) and 20 pmol of each primer. The reaction mixwasoverlaid with25 pi of mineral oil (Sigma). The 'Hot Start' techniquewas used to initiate the reaction: during the first dena- turationstep,the dNTPswereadded through the oil layer of each tube. The final reaction volume was 25 pi.
The samples were amplified at the following temperatures: initial denaturation at 94°C for 3 minutes, then 10 cycles of 1 minuteat94°C (dena- turation), 50 secondsat52-60°C (annealing) and 30 seconds at72°C (extension). In the following 30 cycles the extensionstep was45seconds, and in the last 10 cycles itwas 1 minute. In the last cycle the
samples were held at 72°C for an additional 5 minutes.
After PCR amplification, 10pi from each sample was digested with suitable restriction endonucleases. The ZFY/ZFX product from mice was digested after the addition of 4 IU of Haelll and 10 pi of 10 mM Tris-HCI (pH 7.5), 18.5 mM MgCljand 50 mM NaCl (total reaction volume 20.4 pi). Sheep amplification productswereadded to 5 1U of Sad in 10 pi of buffer A (Boehringer Mannheim). Cattle PCR products were incubated with 5 IU Pstl in 10 pi of buffer H (Boehringer Mannheim). All digestionswereperformed in 37°C for 60-90 min, followed by 15 min at 65°C to enhance the separation of the products. DNA frag- mentswereseparated by electrophoresis (3 %aga- rosestained with 0.5 pg/ml ethidium bromide). The fragmentswerevisualized and photographed under UV illumination. In the mouse, Haelllcutswithin the ZFX but notZFY,yielding twofragments from females (approximately 200 and 250 bp), whereas in males the uncut ZFYproduct results in a third band,about450 bp in size (J.F. Medrano,personal communication). In the sheep the ZFY homologue remains uncut withSad, while the ZFX homo-
logue is digested. Males exhibit three fragments, sized 173,272 and 447 bp, while females lack the largestone(Aasen and Medrano 1990). Sad also digests mouse DNA in a similar pattern. In the bovine, the ZFY product is digested but the ZFX homologue isnot.Consequently, males show three bands (103, 344 and 445 bp) and onlyone(445bp)
isseen in females (Aasen and Medrano 1990). All assays included male and femaleDNA samplecon- trols (extracted from blood) and ablank control.
Results
DNA extracted from tails of micewassuccessfully amplified when 54°C was used for primer annealing, but no detectable amplification was observed using 60°Casthe annealing temperature.
Likewise, DNA from mouse, sheep or cattle em- bryos yielded aPCR product of the expected size using the lower annealing temperature. Biopsies
were also successfully amplified, often even when they consisted ofonesingle cell. Althoughsuccess- ful amplification was obtained using 52°C for primer annealing, 54°C appeared to give a better amplification yield.
Enzyme digestion of the PCR products yielded fragments of the expected size (Fig. 1 and 2). In the sheep, afew of the female samples showeda weak band sized approximately 450 bp. This was dueto incomplete digestion, which could be confirmed after repeated enzyme incubation of such samples.
In bovinemales, the 103 bp fragmentwas weaker than thetwolonger fragments,ascould be expected because of the shorter length. Thus the appearance ofa 344 bp fragmentwasthe clearest indication of maleness. Inmostcasesitwaspossible todiagnose thesexfrom samplesassmallas1-4cells,although the signals in such samples were usually weaker than those obtained from halfor quarterembryos.
Accuracy testingwasdone with sheep embryos, most of whichwereof poor quality. When theem- bryo manipulation and the lysis of samples were
done under farmconditions, 21 replicated samples weresuccessfully amplified from 14 embryos after splitting into 2-4 portions. No replicates were obtained from8 embryos. Of all the samples, 67 % (41/61) could be sexed. All replicates fromagiven embryo yielded identical bandingpatterns.
When all proceduresweredone in the laboratory, total amplificationsuccess was68%(28/41) when the sampleswerelysed in proteinase K. With alka- lictreatmentthesuccessrate wasonly 32%(7/22).
One misidentification of 15 replicates from 9em- bryoswas observed. This contaminationwas most likely due tothe useof thesamemicropipette for handling all the samples in the laboratory. On the farm, a separate micropipette was used for each sample.
Efficiency (proportion of diagnosable samples) dependedonfactors suchasbiopsy size,useof sili- conized pipettes, etc. Larger biopsies were more likelytoyield detectableamountsof the amplified product. Theuseof siliconized pipettes appearedto decrease the attachment of embryostothe pipette, thereby improving the efficiency.
Agric.Sei.Finl. 1(1992)
Discussion
There are some reports describing livestock em- bryo sexing based onPCR (Herretal. 1990a,b, Schröder et al. 1990, Bredbacka et al. 1991, Peura et al. 1991, Setiabudi and Gustafsson 1991), but detailed information is often lacking.
This is no doubt partly due to the commercial
potential of sexing.
In this paperathorough protocol is outlined for sexing bovine and ovine embryos. Although sheep embryo sexing results have been published previ- ously (Herr et al. 1990 b), this paper is to the authors' knowledge the first to describe in detail PCR-based sexing of embryonic cells in this spe- cies.
With the protocol presented in this paper, successful amplification could be obtained fromas few as 1-4 blastomeres. This makes it possible to transfer sexed embryos from which only small biopsies have been removed in order tomaintain high post-transfer viability. In a recent study (Peippo et al., in preparation) 11 of 13 bovine samples (85 %) could be sexed with thepresent method when the biopsy sizewasabout25 %ofa morulastage embryo. In the same study, sex was confirmed using another PCR-based sexing method with all embryos (10/10) yielding identical results.
In themouse,two fetuses which were sexedatthe preimplantation stage wererecovered bycaesarean section,both being correctly diagnosed (Kananen, in preparation). Thus, the method appears to be highly accurate, and with proper handling of bi- opsiesmostsamplescanbe diagnosed.
Fig. 1.Banding patterns fromdigestedZFY/ZFX PCRproducts of male (lanes2,3,7) and femalesheep embryo biopsies (lanes 4, 5,6). Samples inlanes2-7werefrom5,3,7and3 blastomeres,aquarterembryo, anda halfembryo, respectively. Samples 6and 7were preparedunder farm conditions. Lanes 1and 8, molecularweightmarkers.
Fig. 2. Bandingpatterns from bovine male (lanes 1and 3) and female(lanes 2and4)ZFY/ZFX PCRproducts. Male samplesincluded 10ng(lane 1)and 1ng(lane 3)ofDNA.
Both female patternsarefromtwo-cell-stage embryos.Lane 5,molecularweightmarker.
Since the ZFYlocus isnot thetestisdetermining gene,there isaminor risk of chromosomalrearran- gements leading to ZFY-positive females or vice versa.Cautionmustbe exercisedtoavoid contami- nation of anysourceofplacental mammalianDNA, since the distinction betweensexesis dependenton species-specific restriction enzymes. Forinstance, if murine samples are contaminated with bovine DNA, females will be identified as males, since bovine DNA remains undigested with Haelll.Also, fetal calf serum (or bovine serum albumin) may containtrace amountsof DNA resulting in amplifi- cation products. Preliminary trialssuggest that this problem canbe overcomeby exposuretoUV light (Peippo and Bredbacka, unpublished). However, the results presented in this paper would suggest that sample contamination isnot amajor problem.
Compared to the approaches involving control primers, the time required for restriction enzyme digestion is adisadvantage of the protocolpresent- ed in this paper. However, in this study we also examined a faster technique to lyse embryonic cells,utilizing alkalinetreatment at65°C. This che- mical approach saves aboutone hour comparedto the enzymatic method, but needs further optimiza- tion since only aboutonethird of the samples yield- ed amplification products in this study.
With the presented protocol, embryo sexingcan be performed in about five hours when alkaline lysis of samples is utilized. With fastertemperature cycling it is very likely that the protocol can be shortenedeven more, thus making embryo sexing under field conditionsmorepractical.
Since the primers have auniversal nature,initial testingcan be performed with mouseembryos. It is quite possible that different laboratories may need
toadoptsomeminor modifications ofthisprotocol.
Therefore the possibility of usingmouse embryos in the optimization process is an advantage, par- ticularity when the sheep embryo sexing protocol is being optimized, since both species can be sexed with thesame protocol. It may be possibleto test the conditions with blood or any other source of DNA, but thereare some limitations tothis. For instance, the control of the amount of template is better with embryosorembryonic cells than witha highly diluted DNA solution, which atvery low concentrations may totally lack the template sequence. Furthermore, embryonic cells appearto be more resistant to lysis than many other cell types.
Basically embryos from any mammalian species could be sexed by the method presented in this study, provided that sequence differences exist between the X- and Y-chromosomal products. In ideal situations these differencesarerecognized by restriction enzymes. Using a variety of enzymes, Aasen and Medrano (1990) showed RFLP differ- encesbetween thesexes in cattle,sheep, goatand humans,but the RFLPswere identical between the sexesin thehorse,pig and rainbowtrout.However, polymorphisms thatremain unobserved after RFLP analysis could be detectedonthe basis of differen- tial hybridization of allele/homologue specific oli- gonucleotides. This would require sequence infor- mation of the ZFY and ZFX loci.
Acknowledgements. The authors wish to thank Johanna Viikkiforsynthesizingtheoligonucleotidesand Riikka Vel- mala for extracting DNA from the blood samples. The Finnish-Egyptian sheep breeding project provided all the sheep embryosusedinthisstudy.
References
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Manuscriptreceived January1992 Peter Bredbacka
JaanaPeippo
AgriculturalResearch Centre of Finland Departmentof AnimalBreeding SF-31600Jokioinen,Finland
SELOSTUS
Lampaan janaudan alkioidensukupuolilajittelu ZFY/ZFX-lokuksen DNA:n entsymaattisella monistuksella
Peter Bredbacka ja JaanaPeippo
Maatalouden tutkimuskeskus
Lähiaikoina onmahdollista saada haluttuasukupuoltaolevia jälkeläisiä käytännön kotieläintuotannossa. Koska siittiöi- den lajittelu X- ja Y-siittiöihinennenkeinosiemennystä ei vielä ole tuonut toivottavaa tulosta, on ainoa vaihtoehto tehdä määritys suoraan alkiosta ennen alkionsiirtoa.
Alkiosta otetaannäytepala, jonkaDNA:sta määritys voi- daan tehdä. Sukupuolilajittelutehdään useinDNA:n spesifi- sellä monistuksella (PCR)käyttämällä Y-kromosomispesi- fisten alukkeiden lisäksi autosomaalisia alukkeita, jotka vähentävätvirhemääritysten todennäköisyyttä.Tällöin reak- tio-olosuhteiden optimoiminen muodostuu hankalaksi.
Lisäksikäytetytalukkeet ovatyleensä lajispesifisiä,minkä johdosta optimointionnistuu vainkäyttämällä kyseisen lajin DNA:ta. Tässä tutkimuksessanämä ongelmatovatpoistettu monistamalla evoluutiossa konservoitunutta ZFY-geeniä.
ZFY-geeni on kaikkien istukallisten nisäkkäiden Y-kro-
mosomissa,jasilläonhomologiX-kromosomissa.Erilajien sisälläsukupuoltenerottaminen perustuuZFY-geeninsisällä olevien, lajispesifisten pistemutaatioiden tunnistamiseen restriktio-entsyymien avulla. Entsyymi-käsittelyn jälkeen saadaan kunkinlajinnaaraillejakoiraille tunnusomaisetjuo- vastotgeelielektroforeesissa. Menetelmä optimoitiin hiiren DNA:lla, ja sen tarkkuus testattiin pilkkomalla lampaan alkioita 2-4 osaan sekä kenttäolosuhteissa että laborato- riossa. Kustakin osasta tehtiinmääritys erikseen sokkotes- tinä. Samalla verrattiin erilaisia näytteenkäsittelytapojapar- haan tehokkuudenlöytämiseksi. Joissakintapauksissasolu- näytteen entsymaattinen hajotus korvattiin nopeammalla menetelmällä,jossanäyte kuumennettiin emäksisessä liuok- sessa.Käytetyllä tekniikalla määritettiin sukupuoli erittäin suurella tarkkuudella lampaan näytepaloista, jopa yhdestä solusta,sekä naudan 1-8soluisista alkioista.
