Cryoloop vitrification of rabbit oocytes

X.Y. Cai1, G.A. Chen1,2, Y. Lian1, X.Y. Zheng1 and H.M. Peng1

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Vitrification is assumed to be a promising method to cryopreserve human oocytes but still needs optimization. In this study, rabbit oocytes (fertilized by ICSI) were vitrified with cryoloops, and the effect of three different cryopreservation protocols on spindle configuration and embryo quality was assessed. METHODS: Metaphase II rabbit oocytes were randomly assigned to one of four groups: (i) control; (ii) E40 [40% ethylene glycol (EG)]; (iii) ED20 [20% EG + 20% dimethylsulphoxide (DMSO)]; and (iv) ED20 + M (20% EG+20% DMSO+ vitrification machine). After warming, one part of each group was fertilized by ICSI to examine the fertilization and embryo cleavage ability, and the others were immunostained for tubulin and chromatin before visualization using confocal microscopy. RESULTS: The survival rates after warming were 79.1, 83.1 and 82.3%, respectively. In protocols E40 and ED20, the spindles were severely injured and the embryo quality not good compared with those in the ED20 + M group. CONCLUSIONS: The fastest cooling rate in combination with EG and DMSO as cryoprotectants had the fewest adverse effects on the spindle configuration of rabbit oocytes and embryo development.

Key words: cryoloop/cryopreservation/oocyte/rabbit/spindle/vitrification


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Oocyte cryopreservation has the potential to become an important technique for preserving gametes for females whose fertility may become compromised by medical treatment or the physiological effects of ageing. It could be helpful in donor oocyte programmes and an attractive alternative to embryo cryopreservation. It has gained more attention (Gook and Edgar, 1999Go; Paynter, 2000Go; Kuleshova and Lopata, 2002Go; Van der Elst, 2003Go) since ICSI was used to solve the fertilization problem of frozen oocytes (Gook et al., 1995Go; Kazem et al., 1995Go). Over one decade after the first pregnancy was achieved using frozen and thawed oocytes (Chen, 1986Go), Fabbri reported a modified slow freezing and rapid thawing method to freeze human oocytes that gave higher rates of oocyte survival and embryo development (Fabbri et al., 2001Go) than before (Porcu et al., 1997Go; Polak de Fried et al., 1998Go; Fabbri et al., 1998Go; Tucker et al., 1998Go; Yang et al., 1999Go). However not all centres have obtained satisfactory results (Chen et al., 2002Go; Boldt et al., 2003Go; Fosas et al., 2003Go). Vitrification can be used to freeze mammalian oocytes and embryos (Hong et al., 1999Go; Kuleshova et al., 1999Go; Lane et al., 1999Go; Chung et al., 2000Go; Yoon et al., 2000Go). Vitrification is simple and rapid compared with slow freezing; however, it still needs more studies, including animal research.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Oocyte and sperm collection
Mature Japanese white female rabbits were superovulated by six successive subcutaneous injections of FSH (0.15 mg/each, Institute of Zoology, Academia Sinica) at 12 h intervals followed by 100 IU of HCG (Sigma) 12 h after the final dose of FSH (Li et al., 2001Go). The oviducts were flushed with Earle's balanced salt solution (EBSS; Sigma) containing 10% fetal calf serum (FCS; Hyclone) 14–15 h after HCG injection. The cumulus cells were removed from the oocytes by mechanically pipetting after exposure to 300 IU/ml hyaluronidase (Sigma). The corona radiata cells were not removed. After washing 3–5 times in EBSS containing 10% FCS, the oocytes were then transferred to medium 199+10% FCS for freezing.

Rabbit semen was collected from mature Japanese white male rabbits using an artificial vagina and washed 3–4 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 5.5–6 µm inner and 7.5–8 µm outer diameter. The micromanipulation method was as described by Li et al. (2001)Go. 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 5–7 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., 1999Go), albeit with slight modifications. The cryoloop consisted of a nylon loop (20 µm width; 0.5–0.7 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 3–5 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 3–5 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 4–6 times in culture medium and then incubated at 37 °C, 5%CO2. ICSI was done ~2 h later within a period of 1–1.5 h.

Fixation and immunocytochemical labelling
Oocytes were stained using a modified protocol already described by Baka et al. (1995)Go. 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-{alpha}-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 argon–krypton laser (TNS SP2, Germany). When FITC fluorescence was monitored, the wavelength of excitation light was 488 nm and the wavelength of emission light was 515–535 nm. When PI fluorescence was monitored, the wavelength of excitation light was 543 nm and the wavelength of emission light was 590–630 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 {chi}2 analysis. Differences were considered significant when P was ≤0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Oocytes (n=2070) retrieved from superovulated rabbits were randomly distributed in four groups. Results for the survival after warming, ICSI, fertilization and embryo cleavage rates in the different experimental groups are summarized in Table I. A total of 187 rabbit oocytes were vitrified by the E40 protocol (group 2), with 148 intact upon warming, resulting in a survival rate of 79.1%. However, only six of them survived after ICSI, and none of them was fertilized. A total of 503 rabbit oocytes were vitrified by the ED20 protocol (group 3), with 418 (83.1%) surviving after warming and 58 (15.3%) surviving after ICSI, 27 (46.6%) were fertilized and 11 (19.0%) cleaved. In the ED20 + M group (group 4), 887 (82.3%) of 1078 oocytes survived after warming, 84 (10.0%) survived after ICSI, 63 (75.0%) were fertilized and then 31 (36.9%) cleaved. No significant differences were observed in survival rate after warming among the three groups. The survival, fertilization and cleavage rates in group 3 and group 4 after ICSI were significantly lower than those in the control group (group 1) (15.3 and 10.0% versus 78.5%; 46.6 and 75.0% versus 83.0%; 19.0 and 36.9% versus 63.9%, respectively, P<0.001). The survival rate after ICSI in group 3 is a little higher than that in group 4 (15.3 versus 10.0%, P<0.01), but the fertilization and cleavage rates in group 3 were lower than those in group 4 (46.6 versus 75.0% and 19.0 versus 36.9%, respectively).


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Table I. Survival, fertilization and cleavage rate in the four experimental groups

 
The embryo qualities after ICSI are listed in Table II. No significant differences in symmetrical embryo cleavage rate were observed (89.8 versus 83.9%, respectively) between the control group and ED20 + M group, but it was lower in the ED20 group (27.3%).


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Table II. Proportion of embryos undergoing symmetrical cleavage

 
The spindle configurations were revealed by an immunofluorescent method in 26 oocytes from the control group, 20 from group 2, 22 from group 3 and 35 from group 4. The spindle structures were all located at the periphery of the oocyte, and oriented perpendicular to the plasma membrane. Spindle configuration was regarded as morphologically normal when a barrel-shaped structure with slightly pointed poles was formed by organized microtubules traversing from one pole to another. The chromosomal configuration was regarded as normal when chromosomes were arranged on a compact metaphase plate at the equator of the structure (Figure 1). A normal spindle means that spindle configuration, spindle microtubules and chromosomes are all in normal conditions. Abnormalities include disorganization of microtubules, and chromosomes displaced from the plane of the metaphase plate. Details of the abnormal patterns are given in Figures 2 and 3.



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Figure 1. Control group. (AH) Normal development process of rabbit embryos achieved using fresh oocytes fertilised by ICSI; (I) Rabbit oocyte with normal spindle and polar body under confocal microscope after immunostain; (J–K) Normal spindle of rabbit oocyte with chromosomes arrayed at the metaphase plate (under confocal microscope and fluorescent microscope respectively).

 


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Figure 2. E40 group. (A and B) Abnormal spindle configuration with condensed microtubules; (C) Abnormal spindle configuration with condensed microtubules and dispersed chromosome.

 


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Figure 3. ED20 group. (A and B) Asymmetrical cleavage of rabbit embryos with 2 and 4–6 cells; (C and D) Abnormal spindle with dispersed chromosomes; (E and F) Abnormal spindle with disrupted microtubule bundles; (G and H) Abnormal spindle with twisted microtubule bundles.

 
Figures 14 gave the embryo cleavage and spindle conditions of the four experimental groups, respectively. In groups 1 and 4, the embryo cleavage was symmetrical and the spindle figurations were mostly normal. In groups 2 and 3, different abnormal spindle types were observed, while no oocytes were fertilized in group 2 and embryo cleavage resumed asymmetrically in group 3.



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Figure 4. ED20 + M group. (AF) Development process of rabbit embryos achieved using frozen and thawed oocytes fertilitzed by ICSI; (GI) Normal spindle configuration of rabbit frozen and thawed oocytes.

 
The results of the normal spindle rates in three groups are shown in Table III. Statistically significant differences were observed between the control group and the ED20 + M group (76.9 and 62.9%, respectively) and between the other two groups (30.0 and 27.3%, respectively, P<0.01).


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Table III. Proportion of oocytes with normal spindles

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Human oocyte cryopreservation has not been fully optimized. On the one hand, the demand for this technique in clinical work is increasing with the development of reproductive technology, while the progress on studies of it is slow. Up to now, this technique still cannot be routinely applied in clinics. Human oocytes are very precious, so large-scale studies on oocyte cryopreservation using human oocytes are not realistic. Mouse oocyte cryopreservation provided many data. However, the protocol successful with mouse oocytes was inefficient for human oocytes (Chen et al., 2000Go), in part because the diameter of mouse oocytes (~60 µm) is much smaller than that of human oocytes (~130 µm). Since the oocyte volume influences the permeation time and distance of intracellular cryoprotectives, this is a very important factor in cryobiology. The volume of rabbit oocytes (~120 µm) is similar to that of human oocytes. However, ICSI of rabbit oocytes is very difficult, because the rabbit oocytes have rough, dark granules in the plasma, and they easily lyse and die after the ICSI process. However, they are readily activated after ICSI compared with bovine oocytes (~130 µm). Only two studies on ICSI success in fresh rabbit oocytes have been reported (Deng and Yang, 2001Go; Li et al., 2001Go).

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., 1994Go; Songsasen et al., 1995Go). 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., 1991Go; Bos-Mikich and Whittingham, 1995Go). 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., 1994Go). 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 4–24 °C (Pickering and Johnson, 1987Go), 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 frozen–thawed mouse oocytes by slow and ultra-rapid freezing, concluding that the ultra-rapid freezing protocol preserved spindle integrity better (Aigner et al., 1992Go). 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., 1999Go). Apart from the reassurances given in a few reports (Gook et al., 1994Go; Cobo et al., 2001Go), 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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 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on October 19, 2004; resubmitted on December 14, 2004; accepted on January 19, 2005.





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