Journal of Assisted Reproduction and Genetics, Vol. 16, No. 3, 1999

SHORT COMMUNICATION

CHICAGO, ILLINOIS

Visualization of Chromosomes in SingleHuman Blastomeres

The present work describes our results fromemploying these techniques for visualization of thechromosomes from single human blastomeres andapplication of this method for the detection of translo-cations.

Submitted: December 16, 1998Accepted: December 23. 1998

INTRODUCTION

Preimplantation genetic diagnosis (PGD) makes itpossible to select genetically normal embryos forpatients at risk of having children with genetic andchromosomal disorders. The major limitation of thistechnique is the amount of material available forgenetic analysis. It usually involves either the first andsecond polar bodies or only one or two blastomeresbiopsied at the cleavage stage (1,2). FISH analysis ofthe first polar bodies has been used to detect chromo-somal translocations in human oocytes (3,4). However,this technique is limited for maternally derived translo-cations and since, at present, there is no reliable methodfor visualizing blastomere chromosomes, paternallyderived translocations cannot be detected at the preim-plantation stage.

Nuclear transplantations have been extensively usedfor the study of nucleocytoplasmic interactions, celldifferentiation, and animal and embryo cloning(reviewed in Ref. 5). It is usually expected that heterol-ogous nuclei, if transferred into enucleated eggs andzygotes, would undergo remodeling in accordance withthe developmental program of the ooplasm. In thisstudy, we investigated the possibility of applyingnuclear transplantation techniques for karyotyping asingle blastomere of preimplantation human embryo.Our expectations were based on the fact that individualblastomeres, when fused with ooplasts, will beinvolved in the cell cycle, characteristic for recipientooplasm and thus, the timing of mitosis could bemanipulated and predicted.

MATERIALS AND METHODS

The use of human oocytes and preimplantationembryos for this project was approved by IRB of Illi-nois Masonic Medical Center, Chicago, IL. The useof hamsters was approved by ACC of the Universityof Illinois at Chicago. Frozen mouse zygotes werepurchased from Charles River Laboratories (Wilming-ton, MA).

Unless stated otherwise, all chemicals and reagentswere purchased from Sigma Chemical Co. (St. Louis,MO). Embryo culture and micromanipulations wereperformed in HTF medium with 10% Plasmanate(Bayer Biological, New Haven, CT). Micromanipu-lations were performed under a Nikon Diaphot micro-scope equipped with a warming stage, Hoffmanmodulation contrast optics, and epifluorescence forHoechst stain. The double-instrument micromanipula-tion technique, initially proposed by Tsunoda et al.(6), was used for oocyte-zygote enucleation and forblastomere biopsy. MII oocytes not fertilized after IVFor ICSI, or MII oocytes matured for 24-48 hrs in vitrofrom immature oocytes (Germinal Vesicle or MI) wereenucleated and served as ooplast recipients. Prior toenucleation, oocytes were incubated for 10-15 min at37°C in medium with 1 jjig/ml Cytochalasin D, 0.2(jig/ml Demecolcine, and 0.5 fjig/ml Hoechst 33342.Enucleation procedure was basically the same asdescribed by Tsunoda et al. (7) for mouse oocyte enu-cleation.

Enucleation of abnormally fertilized zygotes wasachieved by first exposing 1- or 3-pronuclear zygotesfor 10 min to a medium containing 1 (Jig/ml Cytocha-lasin D and 0.2 |xg/ml Demecolcine. Then zygoteswere transferred for 3-5 min into the medium con-

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134 SHORT COMMUNICATION

taining 0.05 M sucrose, 1 (xg/ml Cytochalasin D, and0.2 fig/ml Demecolcine. Enucleation was performedin the same medium (Fig. 1A and B).

Prior to blastomere biopsy cleavage-stage embryoswere incubated in the medium containing 0.05 Msucrose. Biopsy was performed in the same medium.To reduce the chances of blastomere damage, blasto-mere biopsy was performed using a pipette with ainternal diameter of approximately 40 |xm.

Electrofusion was induced with the aid of the elec-trofusion apparatus (Bams Manufacturers Inc, Chi-cago, IL) in a fusion chamber consisting of twoplatinum wire electrodes glued to the bottom of a glassdish with a gap of 0.4 mm (Fig. 1C). Electrofusionmedium consisted of 0.3 M Mannitol, 0.1 mM MgSO4,0.05 mM CaCl2 and 0.5% polyvinylpyrrolidone. Cellfusion was induced with a single DC pulse at 40 Vfor a duration of 500 JJLS. Cell fusion was assessed 30min after electrofusion (Fig. 2D).

Premature chromosome condensation (PCC) wasinduced by cell exposure to 5 jxM of okadaic acid (OA)diluted to the final concentration with PBS containing 3mg/tnl BSA and 0.5 |xg/ml Cytochalasin D (8). Theoptimal time for inducing PCC in one-pronuclearzygotes was 1 hr.

Blastomere arrest at the metaphase of cleavage divi-sion was achieved by 5-15 hr blastomere or wholeembryo incubation in the culture medium containing0.1 (Jig/ml Demecolcine, or 3 (xg/ml Nocodazole (9),or 0.04 (Jig/ml Podophyllotoxin and 0.04 jxg/ml Vin-blastine(10).

Immediately before fixation, zonae pellucidae wereremoved by Acid Tyrodes solution; Dyban's techniqueof oocyte fixation was used ( 1 1 ) . Chromosome plateswere first assessed using phase contrast and then proc-essed for the whole chromosome painting (WCP).Vysis probes (Vysis, Inc., Downers Grove, IL) andstandard protocol suggested for hybridization and slidewashing were used for the WCP. Slides were imagedwith a Nikon Microphot-FXA microscope equippedwith CE 200A CCD camera (Photometries Ltd.).Quips™ Genetics Imaging Software (Vysis, Inc.) wasused for the chromosome imaging.

RESULTS AND DISCUSSION

Our attempts to obtain metaphases by incubatingblastomeres in the presence of the inhibitors of micro-tubuli (Nocodazole, Demecolcine, Vinblastine, andPodophyllotoxin) supported the previous observation

Fig. 1. Nucleocytoplasmic hybrids between enucleated haploidzygote and 1/8-blastomere. (A, B) Zygote enucleation (X200;reduced at 90% for reproduction), (C) cytoplast and blastomereelectrofusion (X50; reduced at 90% for reproduction), (D) embryos1 hr after fusion (X200; reduced at 90% for reproduction).

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Fig. 2. Three metaphases obtained from a single eight-cell embryo, carrier of unbalanced, maternally derivedchromosomal translocation. Whole chromosome painting (WCP). Chromosome 11 painted orange, chromosome14 painted green, cemromeric region of chromosome ! I stained aqua. Note the t iny telomeric region ofchromosome 14 (green) translocated onto chromosome 11. (A) Metaphase (original magnification X600), (B)prometaphase (original magnification X600). (C) a metaphase (magnifications: metaphase plate at X200, WCPinserts at X600).

(12) that the standard technique of cell karyotyping isnot applicable for PGD.

Okadaic acid, a potent inhibitor of protein phospha-tases 1 and 2A, has been widely used to induce PCC inboth somatic and embryonic cells (13-15). Blastomereincubation in the presence of OA resulted in 20 meta-phases from 89 individual blastomeres (Table I). Thesuccess rate of this technique was too low for use inPGD cases. In most cases, OA treatments had either nonoticeable effect on the interphase nucleus, or inducednucleus picnotization, or have led to chromatin pulveri-zation (characteristic for PCC of the nucleus in the S-phase of the cell cycle; see Ref. 16).

Our results on blastomere fusion with enucleatedhuman and intact hamster oocytes are presented inTable I. Out of 29 nuclear transplants, ooplasm (with

or without additional treatment by OA) induced PCCin 23 nuclei, 16 metaphases with discernable chromo-somes were obtained. In order to avoid the (rather timeconsuming) necessity for oocyte enucleation, we haveattempted to use hamster oocytes as recipients forhuman blastomere nuclei. The result was clearly nega-tive: hamster ooplasm has never induced PCC in thenucleus of the fused human blastomere. Further experi-ments show that, unlike human oocytes, hamsteroocytes are activated by the electric impulse used foroocyte-blastomere fusion. Instead of inducing PCC inthe introduced blastomere, hamster ooplasm led itsnucleus into the beginning of a cell cycle. As a result,when such embryos have been treated with OA 2-3hr after fusion, morphology of the introduced nucleuswas typical for PCC of a nucleus in the S-phase of

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Table I. Analyzable Metaphase Plates Obtained After BlastomereFusion with Heterologous Cytoplasm, as Compared to Blastomere

Treatment by Okadaic Acid (OA)

Number ofblastomeres

8929

23

16

Cytoplastdonors

noneenucleated

humanoocytes

intact hamsteroocytes

enucleatedhumanzygotes

OAtreatment (hrs)

1-30 or 1

0 or 1

1

Number ofmetaphases

2016

0

14

the cell cycle. From the practical point of chromosomepainting, such nuclei are of little value.

The results of human blastomere fusion with enucle-ated human oocyte show that about 45% of the nucleicannot be transformed into analyzable chromosomesby the techniques of PCC induction. Since the embryonuclei have been randomly picked from their fourthcell cycle, this figure reflects the percentage of nucleiin the S-phase, when PCC results in pulverized, notanalyzable chromosomes. This leads to the conclusionthat before attempting to induce PCC in any givennucleus, it should be led into G2-phase. Blastomerefusion with an oocyte, followed by ooplasm activationand in vitro culture, is a well-proven method to guidethe blastomere nucleus into G2 and subsequently intometaphase of the cleavage division. However, unlessblastocyst transfer is considered, this technique isincompatible with embryo transfer in the same cycle.

The use of zygote cytoplasm seems to be moreappropriate for the IVF time frame. As seen from TableI, out of 16 blastomeres analyzed by this technique,14 metaphases, one prometaphase (analyzable byWCP, see Fig. 2B), and one nonanalyzable pulverizedchromosome plate, characteristic for S-phase PCCwere obtained. The technique seems to be compatiblewith the routine IVF time scale:

Day 3, noon: enucleation of 1PN or 3PN humanzygotes (fresh or, if unavailable,frozen-thawed);

14:00: biopsy of single 1/8 blastomeres,which are immediately insertedinto perivitelline space of enucle-ated cytoplasts;

16:00: blastomere-cytoplast fusion;23:00: embryo incubation in OA;

24:00: embryo fixation, probes applica-tion for WCP;

Day 4, 08:00: slides washing, slides imaging;11:00: embryo transfer.

This technique was applied for confirmation of aPGD case of maternally derived translocation t(1 1,14),determined by the first polar body analysis (unpub-lished data). Three blastomeres of the embryo pre-dicted to have unbalanced translocation, which was nottransferred, were fused with zygote cytoplasts. Eighthours later, hybrids were transferred into OA and fixed1 hr later. Two metaphases and one prometaphase wereobtained, all showing the presence of unbalanced trans-location, thus confirming the first polar body diagnosis(Fig. 2).

Our preliminary attempts to use frozen-thawedmouse zygotes as cytoplast donors for human blasto-meres gave promising results. The next day, afterfusion, three hybrids were at the pronuclear stage andeight hybrids cleaved and had two nuclei, indicatingthat the metaphase of the first cleavage of such hybridscan be obtained.

The proposed technique provides the possibility forobtaining metaphase chromosomes from human blas-tomeres. However, its application for human PGD willdepend on the availability of human abnormally fertil-ized zygotes, fresh or frozen-thawed, which might bereplaced by commercially available frozen mousezygotes as a source for zygote-stage cytoplasts.

REFERENCES

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2. Hardy K, Martin KL, Leese HJ, Winston RM, Handyside AH:Human preimplantation development in vitro is not adverselyaffected by biopsy at the 8-cell stage. Hum Reprod 1990;5:708-714

3. Durban M, BenetJ,Sarquella J, Egozcue J, Navarro J: Chromo-some studies in first polar bodies from hamster and humanoocytes. Hum Reprod 1998:13:583-587

4. Munne S, Scott R, Sable D, Cohen J: First pregnancies afterpreconception diagnosis of translocations of maternal origin.Fertil Steril 1998;69:675-681

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8. Verlinsky Y, Evsikov S: Karyotyping of human oocytes bychromosomal analysis of the second polar bodies. Mol HumReprod 1999 (in press)

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13. Steinmann KE, Belinsky GS, Lee D, Schlegel R: Chemicallyinduced premature mitosis: Differential response in rodent andhuman cells and the relationship to cycl in B synthesis andp34 (cdc2)/cyclin B complex formation. Proc Natl Acad SciUSA 1991:88:6843-6847

14. Dyban AP, De Sutler P, Dozortsev D, Verlinsky Y: Visualizationof second polar body chromosomes in fertilized and artificiallyactivated mouse oocytes treated with okadaic acid. J AssistReprod Genet 1992;9:572-579

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Sergei EvsikovYury Verlinsky1

Reproductive Genetics InstituteChicago, Illinois

1 To whom correspondence should be addressed at ReproductiveGenetics Institute, 836 West Wellington Avenue, Chicago, Illi-nois 60657.

Journal of Assisted Reproduction and Genetics. Vol. 16, No. J. 1999