Department of Obstetrics and Gynecology, College of Medicine and The Hospital, National Taiwan University, Taipei, Taiwan
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Abstract |
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Key words: closed pulled straws/meiotic spindle/mouse oocytes/oocyte vitrification
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Introduction |
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We had examined the effect of vitrification with ethylene glycol (EG)-based cryoprotectants on human oocytes in conventional straws, and found a high survival rate after dilution (Chen et al., 2000a). The morphological survival rate appeared to be greater than that of the slow freezing method reported in the literature (Tucker et al., 1998
). The rates of normal fertilization and early cleavage of vitrified oocytes were comparable with those of the controls (Chen et al., 2000a
). However, the subsequent development and blastocyst formation seemed impaired. Even so, considering the high rate of survival and the early development of vitrified oocytes, further studies were warranted to look for ways to improve developmental capacity.
Martino and colleagues found that chilling damage occurred very quickly with bovine oocytes and that this could affect the developmental potential. To obtain more rapid cooling and warming rates, they put oocytes on electron microscope grids for vitrification, and found a higher percentage of blastocyst formation than that achieved using conventional straws (Martino et al., 1996). Vajta and co-workers developed open pulled straws (OPS) to hold bovine oocytes in a very small amount of vitrification solution (Vajta et al., 1998
). They reported that the pregnancy potential of vitrified oocytes was improved, compared with those using conventional straws. The advantages and disadvantages of conventional straws, OPS, or grids for oocyte vitrification deserve further study.
The microtubules of oocytes are vulnerable to cryoprotectants and to temperature change (Pickering et al., 1990; Gook et al., 1993
; Van Blerkom and Davis, 1994
). Damage to the meiotic spindle may impair fertilization and the development of embryos (Eroglu et al., 1998
). In mouse oocytes, we found that vitrified oocytes exhibited serious disturbances of the microtubules immediately after dilution (Chen et al., 2000b
). After a 1 h incubation, the microtubules could repolymerize so that oocytes vitrified using OPS recovered a significantly higher percentage of normal spindles than those using conventional straws. However, the morphological survival rate with OPS was lower. More research should be done to improve both the survival rate and the microtubules of vitrified oocytes.
Accordingly, we attempted to modify the loading of pulled straws into a closed system, called closed pulled straws (CPS). CPS has the characteristics of OPS as a rapid thermal change method, and of conventional straws as being a non-contact mode. The process of repolymerization of microtubules for cryopreserved oocytes may be time-dependent (Aigner et al., 1992). Whether more spindles of vitrified oocytes can be recovered if they incubate longer post-dilution deserves further investigation. In this study, we explored the effects of vitrification of mature mouse oocytes using CPS, conventional straws, OPS or grids on the morphological survival post-dilution and the meiotic spindles after 1, 2 and 3 h incubations. The capacity of fertilization and embryonic development were examined in vitro.
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Materials and Methods |
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Preparation of pretreatment, vitrification and dilution solutions
The solutions for pretreatment, vitrification, and dilution were prepared using Dulbecco's phosphate-buffered saline (DPBS) (Gibco, Grand Island, NY, USA) plus 20% fetal cord serum. The fetal cord serum was made of heat-inactivated serum of umbilical cord blood that was collected after delivery with the mother's permission. The pretreatment solution contained 1.5 mol/l EG (Sigma). The vitrification solution consisted of 5.5 mol/l EG and 1.0 mol/l sucrose (EG5.5) (Ali and Shelton, 1993). The solutions for dilution contained 0.5, 0.25, and 0.125 mol/l sucrose.
Manufacture of the pulled straws
The 0.25-ml plastic straws (I.V.M., l'Aigle, France) were heat-softened over a hot plate and pulled manually. The pulled straws were cut at the tapered end with a blade. The inner diameter of the tip was 0.8 mm with a wall thickness of ~0.07 mm (Vajta et al., 1998).
Vitrification of oocytes in CPS
The oocytes (46 at a time) were pretreated with 1.5 mol/l EG for 5 min. They were transferred into a drop (200 µl) of EG5.5 on a dish and mixed for equilibration. Then they were transferred to another drop (200 µl). The tip of the pulled straw was loaded with 2 mm of vitrification medium, 2 mm of air, 2 mm of vitrification medium containing oocytes, 2 mm of air, and 2 mm of vitrification medium using a syringe (Figure 1A,B). The vitrification medium containing oocytes was isolated by two small segments of air and medium. Through this closed loading system of CPS, the oocytes will not directly contact with liquid nitrogen, which may occur with OPS. The procedures were performed at a room temperature of 2224°C. The total exposure to EG5.5 lasted for 1 min. They were then plunged into liquid nitrogen for cooling and storage.
After storage from 1 h to 5 days, the CPS was removed from the liquid nitrogen for warming. The opposite end of the pulled straw was sealed using the index finger (Figure 1C). The content was then expelled into a drop of 0.5 mol/l sucrose (400 µl) by using the increase in air pressure in the tube caused by the thermal change. Next, the oocytes were transferred into 0.5, 0.25 and 0.125 mol/l sucrose solutions in a 4-well dish. They spent 2.5 min in each solution. Considering the adverse effect of room temperature on oocytes (Pickering et al., 1990
), we operated the extraction steps in a modified incubator at 37°C. The oocytes were then washed, transferred into the culture medium, and incubated.
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Vitrification of oocytes in OPS
After pretreatment with 1.5 mol/l EG and exposure to EG5.5, loading into the tip of the pulled straw was done through the capillary effect by simply touching a microdrop (12 µl) of vitrification solution containing oocytes (Vajta et al., 1998). Plunging the OPS into liquid nitrogen performed the cooling and storage. At warming, the tip of OPS was put into 0.5 mol/l sucrose solution, and the oocytes were expelled. They were then diluted and incubated.
Vitrification on the electron microscope copper grids
The oocytes were pretreated with 1.5 mol/l EG, and mixed with EG5.5. They were then transferred onto the grid (Agar Scientific Ltd., Essex, England) in very small amounts (Martino et al., 1996). To reduce the volume of vitrification solution, the underside of the grid was blotted on a millicell membrane (Millipore, Bedford, MA, USA). The grids were plunged into liquid nitrogen using fine forceps, and stored there. At warming, the grids were picked up and put into the stepwise dilution solutions. Finally, the oocytes were transferred to culture medium.
Definition of morphological survival
Oocytes were defined as having morphologically survived if the cells possessed an intact zona pellucida and plasma membrane and refractive cytoplasm. They were counted and recorded.
Fluorescent staining of meiotic spindles and chromosomes
The morphologically surviving oocytes of the four treatment groups were examined for spindles and chromosomes after 1, 2 and 3 h incubations. The control oocytes were also tested for comparisons. The procedures for fixing and staining have been described in detail previously (Chen at al., 2000b). In brief, oocytes were preserved in 2% formaldehyde (Merck, Darmstadt, Germany) with 0.02% Triton X-100 (Merck) at 37°C for 30 min. They were then incubated with anti--tubulin monoclonal antibody (Sigma) in DPBS with 0.5% BSA for 45 min and washed in 0.01% Tween-20 (Merck) for 15 min. Tubulin was stained by fluorescein isothiocyanate (FITC) conjugated anti-mouse IgG (Sigma) for 45 min, and Hoechst 33258 (20 µg/ml) (Sigma) stained the chromatin. Excess antibody and dye were washed out in 0.01% Tween-20 for 15 min. The oocytes were transferred into DPBS with 0.5% BSA for 60 min and then wet mounted.
Observation of spindles and chromosomes
Fluorescence was observed using an Optiphot microscope with a magnification of 400x (Nikon, Tokyo, Japan). A Nikon filter of set B-2A for the wavelength of 450490 nm was employed for the FITC green signal of the spindle. A UV-2A filter for the wavelength of 330380 nm was used to search for the Hoechst 33258 blue fluorescence of chromosomes. The microscope was equipped with a digital imaging camera, and pictures were acquired and processed on a computer (Chen et al., 2000b).
Assessment of spindle morphology and chromosome arrangement
Normal spindle morphology was barrel-shaped with microtubules traversing between both poles and chromosomes. The metaphase chromosomes aligned regularly in a compact group along the equatorial plane (Figure 2A,C). Abnormal spindle morphology included a reduction in the number of microtubules or the size (Figure 2B), disruption, or the complete absence of a spindle (Frydman et al., 1997
). Dispersion of chromosomes was defined as abnormal (Figure 2D
).
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Statistics
The morphological survival rates of oocytes in the four treatment groups were calculated. The patterns of the meiotic spindles and chromosomes after 1, 2 and 3 h incubations were analysed for the five groups. The percentages of fertilization and blastocyst formation were compared. A contingency table analysis was performed with several rows and columns for overall difference prior to comparisons between individual groups. If it was significant, 2 or Fisher-exact test was then carried out for comparisons of groups two by two. A P value < 0.05 was considered statistically significant.
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Results |
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Discussion |
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Vitrified oocytes revealed severe disturbances of microtubules immediately post-dilution (Chen et al., 2000b). During the incubation period, the microtubules could repolymerize and start to restore normal spindles. In the present study, we verified that vitrified oocytes recovered more normal spindles after 2 or 3 h incubations, a significant improvement over 1 h incubation periods. In addition, we demonstrated that oocytes vitrified in CPS, OPS, or grids had more normal spindles than conventional straws at 1 h after thawing. The former also had more compact chromosomes than the latter did. Appropriate organization of microtubules is indispensable for the alignment of chromosomes, and disorganization of the spindle may result in chromosome dispersion.
Oocytes held in a pulled straw or on a grid for vitrification achieve a faster cooling and warming rate (a theoretical rate of 20 000°C/min and 180 000°C/min respectively) than those in conventional straws (2500°C/min) (Rall and Fahy, 1985; Martino et al., 1996
; Vajta et al., 1998
). Moreover, oocytes in a small amount of vitrification solution can be directly warmed and immediately diluted into the dilution solution. That reduces exposure to unsuitable temperatures and concentrated cryoprotectants. In contrast, the conventional straw is warmed in water and then cut with scissors. The oocytes in a larger vitrification volume are expelled into the dilution solution and then placed into another dilution solution. This allows more time to pass through the inappropriate conditions (Chen et al., 2000b
). That may explain why vitrification of oocytes using CPS, OPS, or grids preserves spindles better than conventional straws. It may also partly explain the findings of others (Martino et al., 1996
; Vajta et al., 1998
) that the developmental competence of vitrified bovine oocytes could be enhanced using OPS or grids, compared with conventional straws.
Chung and co-workers used grids for vitrifying human oocytes, and found that the culture time before vitrification did not significantly affect embryo development after thawing, but that the best results were obtained in the longest culture period group (Chung et al., 2000). The visual changes in spindles after vitrification were linked with functional effects of oocytes on fertilization and development (O'Neil et al., 1997
). It has been found that insemination of oocytes, cryopreserved by a slow freezing method immediately after thawing caused impairment of fertilization and cytoskeletal dynamics post-fertilization that was improved by insemination after a 1 h incubation (Eroglu et al., 1998
). A sufficient restoration of cytoskeleton after incubation would be critical for fertilization events (Joly et al., 1992
). In our study, the vitrified oocytes at 1 h incubation had significantly lower percentages of fertilization and blastocyst formation than controls. After 2 or 3 h incubations, the fertilization outcome of vitrified oocytes was improved in a way that appeared compatible with the recovery of spindles. The timing of insemination of human cryopreserved oocytes varies in the literature, ranging from 14 h post-dilution (Porcu et al., 1997
; Tucker et al., 1998
; Young et al., 1998
; Kuleshova et al., 1999
). The most appropriate time for insemination may be related to the method of cryopreservation and the recovery of spindles after dilution. This deserves further study.
Achieving faster cooling, warming, and dilution, mouse oocytes vitrified using CPS, OPS, or grids could alleviate the injury of spindles and accelerate their recuperation, compared with conventional straws. The effect on the spindle and chromosome organization for the former three methods appears similar. However, the number of oocytes in each processed group is too low to draw a conclusion. The morphological survival of oocytes using OPS or grids is lower. Therefore, CPS has the advantages of achieving a high survival rate and preserving good spindles. The grid, OPS, CPS, and even the conventional straw for storing the oocytes in liquid nitrogen have a potential risk of infection (Kuleshova and Shaw, 2000). For any clinical application of the method the aseptic condition and avoidance of contamination should be specially planned. Recently, several successful pregnancies from vitrified human oocytes using OPS or grids have been reported in the literature (Hong et al., 1999
; Kuleshova et al., 1999
). Vitrification of human oocytes using CPS regarding the survival, timing of insemination, and potential of pregnancy may deserve further study. The value of CPS for vitrification or ultra-rapid freezing of embryos may also merit investigations.
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Acknowledgements |
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Notes |
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References |
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Submitted on January 8, 2001; accepted on July 28, 2001.