1 Interuniversitäres Forschungsinstitut für Agrarbiotechnologie, Tulln and 2 Ludwig Boltzmann Institute für immuno-, zyto- und molekulargenetische Forschung, Vienna, Austria
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Abstract |
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Key words: biopsy/cryopreservation/mice/micromanipulation/refreezing
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Introduction |
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Depending on which type of genetic analysis is to be used and the time required to perform it, biopsied embryos may require cryopreservation. Human frozenthawed embryos with a reduced number of cells develop and implant normally (Trounson and Mohr, 1983). Cryopreservation of mouse biopsied embryos has been shown to have no adverse effect on their survival and development (Krzyminska and O'Neil, 1991
). However, the conventional freezing protocols have not been found to be optimal for freezing of human (Magli et al., 1999
) and mouse biopsied embryos (Wilton and Trounson, 1989
). The demonstration that embryos can be efficiently refrozen without killing them offers the opportunity to refreeze those carrying genetic defects with the possibility of repairing the defects at some unspecified time in the future. Retrospective analysis of cryopreserved embryos is increasingly attractive as new molecular probes become available to assay specific inherited defects not previously measurable. Although many types of genetic analysis can now be performed within a few hours, the type, sensitivity and reliability of such analyses continue to improve at a rapid pace. Furthermore, many academic institutions and animal breeding organizations have amassed large numbers of cryopreserved embryos in recent years. The capability to analyse such embryos retrospectively, and refreeze them in order to have time to make the most productive use of such embryos, would also increase their value. In-vitro development of refrozen human (Baker et al., 1996
), mouse (Fields et al., 1991
; Vitale et al., 1997
) and bovine embryos (Vitale et al., 1994
; Nowshari and Brem, 1999
) and in-vivo development of mouse intact (Leibo et al., 1991
) and biopsied (Snabes et al., 1993
) embryos has been reported. However, in most of these studies conventional freezing procedures, which are time consuming, require freezing protocols which may not be optimal for freezing of biopsied embryos. Therefore, in this experiment in-vivo development of intact and biopsied embryos using a rapid-freezing procedure, previously found to be optimal for freezing different stages of mouse embryos (Nowshari et al., 1995
; Nowshari and Brem, 1998a
) and oocytes (Nowshari et al., 1994
), was studied.
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Materials and methods |
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For collection of genitalia, flushing and washing of embryos, medium M2 (Whittingham, 1971) with 10% fetal calf serum (FCS; Gibco BRL, Life Technologies, Vienna, Austria) was used. For each repetition, embryos from 46 mice were pooled and washed three times in the drops of flushing media and examined microscopically at x40. Morphologically intact morula-stage embryos were selected and transferred to fresh drops of flushing media. For both experiments, embryos were collected in 12 replicates. Each experiment was repeated 68 times.
Freezing and thawing
The freezing solutions of 1.5 mol/l ethylene glycol (Sigma, Vienna, Austria) with 0.25 mol/l sucrose (Merck, Darmstadt, Germany), 7.0 mol/l ethylene glycol with 0.5 mol/l sucrose and thawing solution of 0.5 mol/l sucrose were made with medium M2. For freezing and refreezing of embryos, freezing solutions were supplemented with 10% heat inactivated FCS. All manipulations were performed in 35 mm plastic Petri dishes (Nunc, Roskilde, Denmark) at room temperature (2223°C).
For freezing and refreezing of embryos, a simple rapid-freezing procedure was adopted (Nowshari and Brem, 1998b). In short, embryos were pre-equilibrated in 1.5 mol/l ethylene glycol with 0.25 mol/l sucrose at room temperature (2223°C) for 5 min. Embryos were then loaded directly with a mouth controlled glass pipette into the middle of a 0.25 ml freezing straw (Mini-tüb, Landshut, Germany) containing 50 µl of ethylene glycol (7.0 mol/l with 0.5 mol/l sucrose). The two ends of the straw were filled with 60 µl of 0.5 mol/l sucrose, which was separated from the medium containing the cryoprotectant by air bubbles. The straws were heat-sealed and immediately (4560 s) dipped, slowly (to avoid cracking) and vertically, into the liquid nitrogen for periods not less than 24 h.
Straws with frozen embryos were thawed in a water bath at 20°C (Nowshari and Brem, 2000b). The cryoprotectants were diluted in a single step by emptying the straws into 0.5 mol/l sucrose solution. Embryos were left in this medium for 5 min and then washed three times in M2.
Embryos (n = 1015) were transferred to 50 µl of M16 supplemented with 4 mg/ml BSA (Crystalline, ICN, Meckenheim, Germany). The droplets of M16 were maintained in an incubator with 5% CO2 in air for 3 h. Embryos were examined for their morphology and either transferred to recipients or refrozen (experiment I) or biopsied/sham-biopsied and refrozen (experiment II). Refrozen embryos were thawed as described above, examined for their morphology and transferred to recipients. Non-cryoprotectant exposed embryos (control) collected in M2 with FCS were left in droplets of M16 supplemented with BSA until their transfer to recipients.
Micromanipulation
The embryos (early morula stage) were biopsied under an inverted microscope (ID 35; Leica, Heidelberg, Germany) equipped with Leitz micromanipulators (Leitz, Wetzler, Germany), attached to a Narishige IM-6 micro-injector. The holding and aspiration pipettes were made from glass capillaries (Hilgenberg, Malsfeld, Germany) on model P-97 Sutter micropipette puller (Sutter, Novato, CA, USA). The holding pipettes had an outer diameter of 6080 µm and an inner diameter of 3040 µm and were polished on the microforge (Bachhofer, Reutlingen, Germany). The pulled aspiration pipettes were cut with the microforge at the place where the outer diameter was 2025 µm. A bevel angle of 30° was made with a microgrinder (Sauer, Reutlingen, Germany). The inner diameter of the aspiration pipette was about 1520 µm. The aspiration pipette was connected to 800 µl Narishige micrometer syringes (Narishige, Tokyo, Japan) by plastic tubing filled with lightweight paraffin oil (BDH; Promochem, Wesel, Germany).
Embryos (n = 1015 at a time) were placed into a drop of medium (M2 without Ca2+ and Mg2+) covered with paraffin oil, positioned on a manipulation chamber mounted on the microscope stage. Embryos were fixed with a holding pipette and the aspiration pipette was forced through the zona pellucida and one blastomere was drawn into the pipette with a gentle suction which was then removed from the embryo. During sham-biopsy, aspiration pipette was forced through the zona pellucida but was withdrawn without removing a blastomere from the embryo.
After the biopsy procedure, the manipulated embryos were transferred to fresh M16 medium and cultured at 37°C in an incubator with an atmosphere of 5% CO2 in air and humidity of 95%. Survival after biopsy was assessed 3 h later under an inverted microscope at a magnification of x100. An embryo was considered to have survived the biopsy procedure if, by inspection under light microscope, all the remaining blastomeres were intact and zona pellucida was not lost.
Biopsied embryos were refrozen immediately after in-vitro culture for 3 h by the same freezing procedure as described above. Refrozen embryos were stored for at least for 24 h before thawing and subsequent transfer into recipients.
Embryo transfer to recipients
Embryos at morula stage were transferred to pseudopregnant recipients. Recipient mice received only (i) unfrozen (control), or (ii) intact frozenthawed, or (iii) biopsied embryos. Embryos were transferred to the uterine horn (810 embryos per oviduct) of C57 BL x CBA-F1 recipients on day 3 of pseudopregnancy, as previously described (Hogan et al., 1986). Pseudopregnancy was induced by mating with proved vasectomized Him-OF1 males. The recipients were either necropsied on day 15 of pregnancy, when the numbers of implantation sites and live fetuses were counted, or were allowed to carry the fetuses to term. Early fetal resorption was confirmed by dipping the uterus in a solution of ammonium sulphate (10%) to make implantation sites visible. Each fetus was examined for size and any visible abnormality.
Statistical analysis
Differences in survival rate, implantation rate and proportion of live fetuses at necropsy between treatment groups were tested for significance with the 2 test. The level of significance was set at 5%.
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Results |
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Six of the recipients which became pregnant after transfer of biopsied embryos were allowed to carry the fetuses to term, while the rest were necropsied on day 15 after transfer. The transfer of biopsied and sham-biopsied embryos to recipients resulted in comparable implantation rates and survival rates of live fetuses (Table I). The six mice allowed to carry the fetuses to term gave birth to 39 normal live fetuses. None of the fetuses born had any macromorphological abnormalities and the sex ratio was normal (1:1 male to female). After mating at 8 weeks of age of six males with 12 females born from biopsied embryos, all the females became pregnant and delivered 84 (43 males and 41 females) live fetuses. All living young of the second generation were also morphologically normal.
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Discussion |
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A comparable morphological survival of mouse embryos frozen twice was reported earlier (Fields et al., 1991; Leibo et al., 1991
; Vitale et al., 1997
); however, in all of these studies conventional freezing procedures were used. A higher proportion of zona damage of embryos frozen twice or three times with conventional freezing procedure, using a small number of embryos, was reported (Vitale et al., 1997
). Intact embryos frozen once or twice with a rapid-freezing procedure in this experiment and transferred to recipients developed to live fetuses, indicating that embryos can be refrozen using this freezing procedure and an acceptable in-vivo survival of embryos can be expected upon thawing and transfer to recipients. Previously the in-vivo development of mouse embryos refrozen with a conventional freezing procedure was reported in only one study (Liebo et al., 1991). The in-vivo survival of twice frozen embryos in that study was comparable to embryos frozen twice with rapid-freezing procedure in this study. Embryo refreezing may find a practical application in embryo transfer programmes in human and domestic animals. Embryos which have been thawed accidentally or which could not be transferred to recipients because of their unsuitability, ascertainable only after thawing, need not be discarded, but rather refrozen and if suitable transferred next time.
After biopsy of once frozenthawed embryos, 89% of biopsied embryos remained intact. This survival rate appeared to be the same between sham-biopsied embryos in experiment II and intact embryos frozen once or twice in experiment I. This indicates that embryos which do not have intact zonae pellucidae can also survive freezing and thawing procedures and can further develop in vitro and in vivo in a similar way to frozenthawed zona-intact embryos. Complete absence of the zona pellucida has been previously shown to affect the survival and development of intact as well as demi-embryos in mouse (Nowshari and Holtz, 1998) and goats (Nowshari and Holtz, 1993
). Poor results have been reported after freezing of mouse embryos with punctured zonae pellucidae using conventional freezing procedure (Wilton et al., 1989). These authors, however, improved the survival and development rate of biopsied embryos with a rapid-freezing procedure using 4.5 mol/l dimethylsulphoxide (DMSO) as cryoprotectant. A comparable survival rate of biopsied and non-biopsied embryos but a reduced developmental rate of mouse embryos which were frozen after biopsy has been reported (Thompson et al., 1995
). That in-vitro developmental potential of biopsied embryos is not influenced by freezing embryos has also been reported by many authors (Roudebush et al., 1990
; Krzyminska and O'Neill, 1991; Liu et al., 1993
). However, these authors just attempted freezing of freshly biopsied embryos. The successful cryopreservation and refreezing of embryos with a punctured zona pellucida could have applications in experimental embryology other than embryo biopsy. It also should be possible to cryopreserve or refreeze mouse embryos after manipulations such as gene injection, embryo splitting, and nuclear transplantation.
The optimal stage of embryonic development at which embryo biopsy can be performed without compromising the developmental capacity is unclear. In this experiment early morula stage embryos were used and intact and biopsied embryos at this stage can be refrozen and biopsied without affecting the in-vivo development of embryos compared with freshly transferred embryos. Removal of a single blastomere at 4-cell (Wilton et al., 1989) and one or two blastomeres at 8-cell stage (Kryzminski and O'Neill, 1991; Liu et al., 1993) does not influence the developmental capacity of embryos in vitro and in vivo. However, impairment of the developmental capacity of embryos at blastocyst stage after removal of trophectoderm cells has been reported (Monk et al., 1988
). Kryzminsky et al. (1990) reported that 8-cell stage mouse embryos are more suitable for embryo biopsy than 4-cell and morula stage embryos. The developmental capacity of frozenthawed biopsied embryos seems to be mainly dependent on the number of blastomeres remaining in the embryos (Krzyminska and O'Neill, 1991; Liu et al., 1993
). Removal of more than four blastomeres from an 8-cell stage embryo resulted in low survival of embryos in vitro and in vivo (Liu et al., 1993
).
Results of this experiment also show that the offspring from biopsied embryos are normal. None of the offspring had any macromorphological abnormality and they developed and reproduced normally, indicating that biopsy did not affect the reproductive performance of the progeny.
In conclusion, the survival and development in vivo of embryos frozenthawed using a simple rapid-freezing procedure is not influenced by freezing them once or twice. An acceptable survival in vivo of embryos subjected to biopsy after one freezing cycle, refrozen and transferred to recipients after thawing, can be achieved. The results suggest that under certain conditions, it may be possible to utilize cryopreservation and re-cryopreservation of embryos in strategies involving screening of embryos for PGD in human and domestic animals and for other logistic reasons.
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Acknowledgments |
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Notes |
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References |
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Submitted on February 11, 2000; accepted on September 7, 2000.