Culture with Matrigel Inhibits Development of Mouse Zygotes
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Journal of Assisted Reproduction and Genetics, Vol. 14, No. 9, 1997 ANIMAL EXPERIMENTATION
Culture with Matrigel Inhibits Development of Mouse Zygotes
K. M. DAWSON,1,4 J. M. BALTZ,1,3-6 and P. CLAMAN2,4
Submitted: April 16, 1997Accepted: May 23, 1997
Purpose: It was reported that Matrigel improved hatching ofmouse blastocysts produced in vitro from F1, hybrid-derivedzygotes. We investigated whether Matrigel would be simi-larly beneficial with outbred strain-derived embryos, whichexhibit a "two-cell" block similar to the developmentalblocks of other species,Methods: Mouse embryo development was assessed with orwithout Matrigel in KSOM medium, which supports thedevelopment of blocking strain zygotes in vitro, and in humantubal fluid (HTF) medium, which normally does not butwhich is used for human IVF.Results: Matrigel severely inhibited the development ofzygotes to blastocysts in KSOM and did not improve culturein HTF. There was no effect on development from the two-cell stage. We were not able to replicate the previous findingof Matrigel's beneficial effect on hatching of F1-derivedzygotes.Conclusions: Matrigel may be a deleterious addition toembryo culture or coculture systems.
1 Loeb Medical Research Institute, Ottawa Civic Hospital, Ottawa,Ontario, Canada.
2 GOAL Program, Ottawa Civic Hospital, Ottawa, Ontario, Canada.3 Human IVF Laboratory, Ottawa Civic Hospital, Ottawa,
Ontario, Canada.4 Division of Reproductive Medicine, Department of Obstetrics
and Gynecology, University of Ottawa, Ottawa, Ontario K1Y4E9, Canada.
5 Department of Physiology, University of Ottawa, Ottawa, OntarioK1Y 4E9, Canada.
6 To whom correspondence should be addressed at Loeb MedicalResearch Institute, Ottawa Civic Hospital, 1053 Carling Avenue,Ottawa, Ontario K1Y 4E9, Canada.
One approach that has been taken to improving thesuccess of embryo culture has been to coculture preim-plantation embryos with other cells, in order to mimicthe in vivo environment of the oviduct, where embryosgrow in the presence of, e.g., oviductal epithelial andfibroblast cells, and are exposed to products of theirsecretion (1,2). During an investigation of a coculturesystem for mouse embryos, Carnegie et al. (3) foundthat embryos grown on a layer of Vero cells wouldhatch from the zona pellucida at a greater rate thanembryos cultured in a simple serum-free medium. TheVero cells were grown on a commercially availableextracellular matrix material, Matrigel. In controlexperiments, however, Matrigel was found to beequally effective with or without Vero cells, implyingthat any positive effect could be attributed to the pres-ence of Matrigel alone. Thus, they proposed that Matri-gel may be a beneficial addition to embryo culturesystems without resorting to the need for coculturingembryos with epithelial cells.
The original work (3) was done using F1 hybrid-derived mouse embryos, instead of random-bred orinbred-derived embryos, which exhibit a phenomenonknown as the two-cell block. The two-cell block ischaracterized by most zygotes failing to develop inculture past the two-cell stage, which is not a problemwith hybrid-derived embryos. Recently, however,defined media such as CZB and KSOM have beendeveloped which allow even blocking-strain mouseembryos to be cultured successfully from zygotes toblastocysts in vitro (4,5).
Because Matrigel had exhibited a beneficial effectupon nonblocking strain embryo development, wewondered if a similar effect would be found for
543 1058-0468/97/1000-0543$12.50/0 C 1997 Plenum Publishing Corporation
KEY WORDS: culture; embryo; in vitro fertilization; Matrigel;media.
544 DAWSON, BALTZ, AND CLAMAN
blocking strain embryos in media which support theirdevelopment. In addition, we wondered whether Matri-gel might improve the development of blocking-strainembryos in media which would otherwise not supporttheir development.
Therefore, we undertook experiments to determinewhether Matrigel improves the development or hatch-ing rate of blocking strain zygotes in KSOM medium,which generally supports their development to the blas-tocyst stage (5). We also investigated whether Matrigelcould relieve the two-cell block of these same embryoswhen cultured in HTF (human tubal fluid) medium,which normally does not support development to theblastocyst stage (6).
MATERIALS AND METHODS
KSOM medium was prepared from stocks asdescribed by Lawitts and Biggers (5). KSOM-Hepesmedium, used for flushing and handling embryos, isidentical to KSOM except that the NaHCO3 is reducedfrom 25 to 4 mM, 21 mM Hepes-free acid is added,and the pH is adjusted to 7.4 at room temperatureusing NaOH. All chemicals used in KSOM andKSOM-Hepes preparations were embryo culture-tested grade or tissue culture grade and were obtainedfrom Sigma (St. Louis) or BDH (Toronto). HTFmedium (7) was obtained from Pharmasciences (Mon-treal). Bovine serum albumin (1.0 mg/ml; Sigma) wasadded to each medium before use. The compositionsof the media used are shown in Table I.
Embryos were obtained from Crl:CF1, BR ("CF1")outbred female mice or C57B16 X Balb/c F1,("CB6F1") F1hybrid mice (5-6 weeks old) matedwith B6D2F1/CrlBR ("BDF") males (Charles RiverCanada, Montreal). Females were superovulated byintraperitoneal injection of pregnant mare serumgonadotropin (PMSG; 5 IU; Sigma) followed 48 hrlater by intraperitoneal injection of human chorionicgonadotropin (hCG; 5 IU; Sigma). Zygotes wereremoved 23-24 hr post-hCG from excised oviductsby flushing the oviducts with KSOM-Hepes mediumcontaining 300 ug/ml hyaluronidase (Sigma), and thezygotes from four to six females pooled. The zygotes,free of cumulus cells, were washed in KSOM-Hepesand distributed into the appropriate treatment groups.Two-cell-stage embryos were flushed from the ovi-ducts 47-48 hr post-hCG, washed in KSOM-Hepes,and distributed into the appropriate treatment groups.
Embryos were cultured in tissue culture dishes (Fal-con No. 3001) for the KSOM vs HTF comparisonexperiments or in organ culture dishes (Falcon No.3037) for experiments where presence or absence ofMatrigel was compared. Single 50-ul drops of mediumwere placed in each dish or organ culture dish welland covered with medium-equilibrated mineral oil(Sigma). The dishes were equilibrated overnight in 5%CO2/air at 37C. Fifteen embryos were placed in eachdrop. Culture was carried out at 37C in 5% CO2/air.
Table I. Composition of Mediaa
a All media contain 1.0 mg/ml BSA, 100 U/ml Na penicillin G,and 50 ug/ml streptomycin sulfate.
b pH adjusted to 7.4 with NaOH.
Matrigel (Collaborative Biomedical Products, Bee-ton Dickinson)-coated organ culture dishes were pre-pared according to the manufacturer's directions andthe procedure of Carnegie et al. (3): Matrigel aliquotswere stored at -20C until needed, then thawed forseveral hours at 4C. They were diluted eight fold withsterile embryo culture-grade water, and 230 ul wasplaced in the center well of each organ culture dish.Each dish was then swirled to ensure an even coatingof Matrigel. The Matrigel was then allowed to gelovernight at room temperature. Two lots (Nos. 902155and 903025) of Matrigel were used; no difference wasobserved between lots.
Journal of Assisted Reproduction and Genetics, Vol. 14, No. 9, 1997
MATRIGEL INHIBITS MOUSE ZYGOTE CULTURE 545
Each replicate consisted of a control group of 15embryos cultured in parallel with one or more treat-ment groups of 15 embryos.
Zygotes. Development was assessed by recording thenumber of embryos which reached the morula stageafter 3 days (72 hr) in culture, the number whichreached the blastocyst stage after 4 days (96 hr) inculture, and the number of blastocysts after 5 days(120 hr) in culture. Hatching was assessed after 5 days(120 hr) and 6 days (144 hr) in culture.
Two-Cell-Stage Embryos. Development was assessedby recording the number of embryos which hadreached the morula stage after 2 days (48 hr) in culture,the number of blastocysts after 3 days (72 hr) in culture,and the number of hatched blastocysts after 4 days (96hr) in culture.
The data from all replicates were pooled for analysis.Thus, the data from each experiment consisted of sev-eral sets, taken on successive days, recording the num-ber of embryos which reached the appropriate stage,or hatched, and those which did not. Each set of dataforms a 2 X 2 contingency table, which was analyzedusing Fisher's exact test. Because there were oftenresponses near-zero, the usually employed asymptoticapproximation methods of probability calculation fail.Therefore, we used exact calculations of probability(StatXact Turbo, Cytel Software Corp., Cambridge,MA).
Two experiments were performed with blockingstrain-derived zygotes. The first tested the effect ofMatrigel presence or absence on embryo developmentin KSOM medium. The second tested the effect ofMatrigel on embryo development in HTF medium, todetermine whether the ususal block to developmentpast the two-cell stage could be released. One furtherexperiment was performed with blocking strain-derived two-cell-stage embryos, in which the effect ofMatrigel on culture from the two-cell stage in KSOMwas tested.
We also performed a set of experiments utilizingCB6F1 hybrid-derived zygotes, replicating the experi-ments reported by Carnegie et al. (3). Zygotes werecultured in HTF medium with or without Matrigel,and the rate of blastoc