1 Department of Obstetrics and Gynaecology, Third Hospital, Peking University, Peking, China 100083
2 To whom correspondence should be addressed. Email: chenguian{at}bjmu.edu.cn
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Abstract |
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Key words: cryoloop/cryopreservation/oocyte/rabbit/spindle/vitrification
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Introduction |
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The present study used rabbit oocytes as an animal model for oocyte vitrification using cryoloops. The effects of different cryoprotectants and cooling rates in the vitrification process on the spindle configurations and embryo (fertilized by ICSI) qualities were estimated.
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Materials and methods |
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Rabbit semen was collected from mature Japanese white male rabbits using an artificial vagina and washed 34 times with phosphate-buffered saline (PBS) by centrifugation at 500 g for 5 min and then suspended for 1 h in 2.5 ml of PBS+10% FCS. Supernatants were mixed with 10% polyvinyl pyrrolidone (PVP; Sigma) to slow down the sperm's vigorous movement before microinjection (Deng et al., 2001).
ICSI and oocyte culture
The injection needle used for rabbit sperm was of 5.56 µm inner and 7.58 µm outer diameter. The micromanipulation method was as described by Li et al. (2001). Briefly, the polar body was at 6 or 12 o'clock and the point of injection at 3 o'clock. An oocyte was penetrated by the injecting micropipette, a small amount of cytoplasm was drawn into the micropipette, and then the cytoplasm together with the sperm was expelled into the oocyte. Immediately after ooplasmic injection, the injecting micropipette was withdrawn quickly, and the oocytes were released from the holding pipette to reduce the intracytoplasmic pressure exerted on the oocyte. All the micromanipulations were conducted at 37 °C on a warm stage. After injection, the oocytes were transferred to M199 medium+15% FCS and cultured at 38 °C, 5%CO2 in air. Pronuclear formation was examined with an inverted microscope 57 h after sperm injection. The oocytes with two distinct pronuclei and a second polar body were considered fertilized.
Vitrification of oocytes
Rabbit oocytes were cryopreserved by three vitrification protocols using cryoloops. They were randomly assigned to one control group (group 1) or one of three vitrification groups.
Group 2: E40 protocol. The cryoloop vitrification method was adopted from previous reports (Lane et al., 1999), albeit with slight modifications. The cryoloop consisted of a nylon loop (20 µm width; 0.50.7 mm diameter) mounted on a stainless steel support rod that was inserted into the lid of a cryovial (Hampton Research, Laguna Niguel, CA). A metal insert on the lid enabled the use of a stainless steel handling rod with a small magnet (Crystalwand, Hampton Research) for manipulation of the loop at low temperature. The oocytes to be vitrified were first placed in base medium including HEPES (Sigma)-buffered medium 199 (Gibco) and 20% FCS supplemented with 20% (v/v) ethylene glycol (EG; Sigma) at 37 °C on a warming plate for 2 min and then transferred into a small drop of vitrification solution made of 40% EG, 10 mg/ml Ficoll70 (Sigma) and 0.65 mol/l trehalose (Sigma) in base medium. The cryoloop was dipped into the solution to create a thin film. A total of 35 oocytes were suspended in the film using a fine pulled glass pipette. The cryoloop was plunged into a cryovial filled with liquid N2. The transfer of oocytes into the vitrification solution and the vitrification process were performed in <35 s.
Group 3: ED20 protocol. The oocytes were first placed in base medium supplemented with 10% dimethylsulphoxide (DMSO; Sigma) and 10% EG at 37 °C for 2 min and then transferred to vitrification solution made of 20% DMSO, 20% EG, 10 mg/ml Ficoll70 and 0.65 mol/l trehalose in base medium. The vitrification process is the same as above.
Group 4: ED20 + M (vitrification machine, Vit Master, Germany) protocol. The Vit Master vitrification machine was used to provide a very high cooling rate (up to 135 000 °C/min) which is very important for the vitrification process. The basic principle of this machine is to avoid the vapourization of nitrogen by applying negative pressure, a vacuum, on the liquid N2, which lowers its temperature to below its boiling point. The evaporative cooling causes the nitrogen to partially solidify, thus creating a nitrogen slush (about 210 °C). Samples immersed in nitrogen slush cool more rapidly because they come into contact with liquid nitrogen sooner than those immersed in normal liquid nitrogen.
Oocytes were placed in base medium supplemented with 10% DMSO and 10% EG at 37 °C for 2 min and then transferred to vitrification solution made of 20% DMSO, 20% EG, 10 mg/ml Ficoll70 and 0.65 mol/l trehalose in base medium. The cryoloop containing 35 oocytes were plunged into nitrogen slush at about 208 °C within 35 s. After vitrification, the cryoloops were inserted into cryovials and transferred to 196 °C liquid nitrogen and then stored in it for several days. The time taken for the last step should also be <35 s.
Warming of oocytes
To warm the vitrified oocytes, the cryoloops were removed from the liquid nitrogen and quickly placed directly into warming solution of 0.5 mol/l trehalose in base medium. After 5 min, the oocytes were transferred sequentially to 0.3, 0.1 and 0 mol/l trehalose in base medium at intervals of 5 min. All the warming process was conducted at 37 °C. After thawing, the oocytes were washed 46 times in culture medium and then incubated at 37 °C, 5%CO2. ICSI was done 2 h later within a period of 11.5 h.
Fixation and immunocytochemical labelling
Oocytes were stained using a modified protocol already described by Baka et al. (1995). Fixation and all subsequent incubations were carried out at 37 °C. Oocytes were fixed for 30 min in a microtubule-stabilizing buffer containing 2.0% formaldehyde, 0.5% Triton X-100 (Sigma), 1 µmol/l taxol (Sigma) in Dulbecco's PBS and then washed three times in a blocking solution of PBS with 2% bovine serum albumin (BSA), 2% powdered milk, 2% normal goat serum, 0.1 mol/l glycine (Sigma) and 0.01% Triton X-100, stored at 4 °C for up to 3 days. Mouse anti-
-tubulin monoclonal antibody (Sigma; 1:250) was incubated with fixed oocytes for 1 h in PBS containing 0.1% BSA and 0.02% sodium azide (Sigma) at 37 °C. Samples were then washed for 1 h by three changes in blocking solution and then incubated further in a 1:50 diluted solution of fluorescein-conjugated goat anti-mouse immunoglobulin G (IgG; Sigma) for 30 min at 37 °C. Following this step, oocytes were washed three times (5 min each time) in PBS containing 0.02% sodium azide and chromosomes were counterstained by propidium iodide (PI; 5 mg/ml, Sigma) for 15 min. After a brief wash in PBS, the sample were immediately sent to the confocal microscope laboratory. The localization of tubulin and chromatin by fluorescein isothiocyanate (FITC) and PI fluorescence was revealed on a laser-scanning confocal microscope provided with an argonkrypton laser (TNS SP2, Germany). When FITC fluorescence was monitored, the wavelength of excitation light was 488 nm and the wavelength of emission light was 515535 nm. When PI fluorescence was monitored, the wavelength of excitation light was 543 nm and the wavelength of emission light was 590630 nm. The 63xoil microscope was used when spindle images were visualized. The images were recorded on a host computer. The image analysis system is Leica TCS-4D confocal software (USA).
Statistical comparisons were carried out using 2 analysis. Differences were considered significant when P was
0.05.
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Results |
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Discussion |
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We tried our best and built this animal model to study the cryoloop vitrification method to obtain some helpful knowledge and experience in cryopreservation of human oocytes. We should point out that this work is the first report on a successful ICSI procedure in cryopreserved rabbit oocytes. Although in the present study the number of cryopreserved rabbit oocytes surviving the ICSI procedure was much lower compared with the control group (10.0 and 15.3% in the ED20 and ED20 + M groups versus 78.5% in the control group), we still can conclude something from these results.
Good survival rates are very important for cryopreservation. In this study, we used three protocols, and all provided good survival rates. We compared the effects of different cryoprotectants and cooling rates in the vitrification method and found that the use of the fastest cooling rate (the VitMaster) and the use of EG and DMSO with Ficoll and sucrose in the cryoprotective solution provided the minimal injury to the spindle and the best quality of embryos.
EG has come into more widespread use because it is tolerated well and penetrates embryos more rapidly than propanediol or glycerol (Rayos et al., 1994; Songsasen et al., 1995
). This reduces the problems caused by osmotic shock and allows direct dilution or transfer of the cryoprotectants. In our study, no warmed oocytes in the E40 group were fertilized successfully, and the spindle injury was severe. This may indicate that the equilibration protocol, the cryoprotectant or dilution protocol were not optimal.
The other two protocols used the combination of EG and DMSO as the intracellular cryoprotectants. DMSO can, under some conditions, harm the spindle or chromosomes, thereby inducing the abnormal development of embryos when it was used alone or combined with other cryoprotective agents (CPAs) (Pickering et al., 1991; Bos-Mikich and Whittingham, 1995
). However, our study showed that when DMSO was combined with EG, trehalose and Ficoll70 in the vitrification solution and a very high cooling rate was used, the spindle and chromosome of the vitrified oocytes were normal. As we know, EG has a good permeating ability, thus DMSO may help in the process of vitrification.
There is a critical threshold value of cryoprotectant concentration that just permits glass formation without crystallization during cooling, but a higher concentration of cryoprotectant is required to prevent devitrification during warming, so the vitrification method needs a relatively high concentration of cryoprotectants. At the same time, exposure of oocytes to very high concentrations of CPAs is known to damage oocytes because of both osmotic and toxic effects. The addition and removal of penetrating CPAs from oocytes create osmotic imbalance across the cell membrane that results in large volumetric changes and injury to the oocyte membrane and cytoskeletal organization (Rayos et al., 1994). This is the main disadvantage of vitrification and may explain why although the spindle configurations of the ED20 + M protocol were normal, the fertilization and development rates of cryopreserved oocytes in this group were still lower than those of the control group. Therefore, more studies about the membrane and cytoplasmic damage are needed to modify the vitrification method.
In the present study, the ED20 and ED20 + M protocol used the same cryoprotectants, while ED20 + M provided better results, which indicates that the faster cooling rate played an important role in this vitrification protocol.
All the three protocols provided good survival rates in this study, while the results of spindle injury and embryo cleavage were totally different. In the E40 group, the condensed microtubules made the spindle lose the normal shape, and no fertilization was observed. In the ED20 group, the microtubules could be seen broken or twisted with or without chromosomes dispersing from the metaphase plate. Some of the rabbit oocytes in this group were fertilized, but the embryo quality was poor. The unsymmetrical embryo cleavage meant that the embryos might be aneuploid. In the ED20 + M group, the normal spindle rate and the fertilization rate were higher, and the embryo quality looked good, but embryo development was not as good as in the control group. Spindles of oocytes were very sensitive to the cooling process. Early studies found that the organization of the microtubule system of mouse oocytes was affected by cooling at 424 °C (Pickering and Johnson, 1987), and returning to 37 °C for at least 1 h could restore some of the oocytes, but not all of them. There was a report comparing the spindles of frozenthawed mouse oocytes by slow and ultra-rapid freezing, concluding that the ultra-rapid freezing protocol preserved spindle integrity better (Aigner et al., 1992
). They also found severely injured types of spindle multipolarity, spindle absence and dislocation of chromosomes, which were not found in this experiment. In spite of an apparent normal fertilization, unbalanced disjunction could occur, resulting in an aneuploid embryo. Aneuploidy could be also one reason for the poor performance in vitro and in vivo of embryos derived from frozen oocytes (Ludwig et al., 1999
). Apart from the reassurances given in a few reports (Gook et al., 1994
; Cobo et al., 2001
), we need large-scale studies stating the chromosomal normality of embryos derived from frozen oocytes. Also, in the human oocyte cryopreservation procedure, preimplantation genetic diagnosis should be part of clinical oocyte cryopreservation programmes.
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Submitted on October 19, 2004; resubmitted on December 14, 2004; accepted on January 19, 2005.
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