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a Department of Animal Science, Cornell University, Ithaca, New York 14850
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The various steps required for cryopreservation (cryoprotective agent [CPA] loading, cooling below 0°C, seeding, cooling to a low subzero temperature, freezing/storage, thawing, and CPA removal) may contribute individually or cumulatively to oocyte damage that in turn decreases fertilization and development rates [6]. Alterations in the cytological components of mammalian oocytes due to procedures required for cryopreservation have been reported for the mouse [7–11], human [12–15], rabbit [16], and cow [17, 18].
Cooling and CPAs alter the arrangement of chromosomes and microtubules in the oocytes of humans [12, 13] and mice [7, 9–11, 19]. Compared to microtubules, microfilaments appear to be less affected by cooling but are altered in different ways by exposure to CPAs. Propanediol (PrOH) exposure causes blebbing in mouse oocytes [19] and leads to depolymerization of actin in rabbit oocytes [16]. Dimethyl sulfoxide (DMSO) can disrupt the actin network in mouse oocytes [8] but has little effect on rabbit oocytes [16].
Freezing effects on oocyte ultrastructure have been demonstrated also. Damage to the plasma membrane, disorganization of the ooplasm, and cracking of the zona pellucida have been observed in human oocytes after freezing [20]. Changes in the ultrastructure of rabbit [16] and mouse [21, 22] oocytes after freezing have been observed as well.
Only a limited number of studies have reported effects of cryopreservation procedures on the cytology of bovine oocytes. Aman and Parks [18] demonstrated that cooling bovine oocytes to 4°C or 25°C resulted in partial or complete spindle disassembly and some chromosome dispersal. Rewarming oocytes to 37°C from 25°C restored normal spindle morphology, but when oocytes were cooled to 4°C and rewarmed, abnormal spindles formed frequently. After freezing bovine oocytes in glycerol, Schmidt et al. [23] observed dilated mitochondria, multiple ruptures in the plasma membrane, and accumulation of numerous small vesicles in the cortical ooplasm.
The present study was designed to determine the effects of cooling, ethylene glycol (EG) exposure, and freezing on the distribution of chromosomes, microtubules, and filamentous actin (microfilaments) in the bovine oocyte and to examine the ability of oocytes to recover from these manipulations.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovaries were collected from a regional abattoir and transported to the laboratory (about 2 h) in Dulbecco's PBS (DPBS; Gibco, Grand Island, NY) containing 0.5% antibiotic-antimycotic (ABAM, containing penicillin, streptomycin, and amphotericin B; Gibco) at ambient temperature. Upon arrival at the laboratory, ovaries were rinsed and held in 0.9% NaCl. Contents of follicles 2–10 mm in diameter were aspirated into a 50-ml conical tube using a 16-gauge needle connected to a vacuum line. Cumulus-enclosed oocytes and other cellular debris were allowed to sediment in the tubes for 10–20 min, then transferred to sterile Petri dishes containing egg-TALP (114 mM NaCl, 3.16 mM KCl, 2 mM NaHCO3, 0.35 mM NaH2PO4H2O, 10 mM sodium lactate, 2 mM CaCl22H2O, 0.5 mM MgCl26H2O, 100 IU/ml penicillin, 10 mM Hepes, 0.1% ABAM, 0.2 mM pyruvate, 0.3% BSA) [24]. Oocytes were recovered from dishes and were rinsed three times with egg-TALP.
Oocytes with at least one layer of compact cumulus cells were rinsed once in maturation medium (Tissue Culture Medium 199 [TCM 199]; Sigma Chemical Co., St. Louis, MO) supplemented with 10% Fetal Clone I (HyClone, Logan, UT; lot #61652012A), 0.2 mM pyruvate, 6.0 µg/ml bovine LH (NOBL Laboratories, Sioux Center, IA), 4.0 µg/ml bovine FSH (NOBL Laboratories), and 1.0 µg/ml estradiol [25]; they were then placed in a Nunclon (Nunc, Naperville, IL) 4-well multidish (50 oocytes and 0.5 ml maturation medium per well). Oocytes were matured for either 20 or 24 h at 39°C in an atmosphere of 5% CO2 in humidified air.
In Vitro Fertilization
Bull semen from one ejaculate was processed and frozen (0.5 ml) by Genex, Inc. (Ithaca, NY). A single straw of semen containing 40 million sperm was thawed in a 37°C water bath for 2 min, and contents were layered on a gradient of 45%/90% Percoll (Sigma) in Tyrode's solution in a 15-ml conical tube, then centrifuged at 340 x g for 30 min. After centrifugation, the supernatant was aspirated, and the pellet was resuspended in 75 µl of sperm-TALP (2.1 mM CaCl22H2O, 3.1 mM KCl, 1.5 mM MgCl26H2O, 100 mM NaCl, 0.29 mM NaH2PO4H2O, 21.6 mM lactic acid, 10 mM Hepes, 25 mM NaHCO3, 50 mg/ml gentamicin, 1.1 mM pyruvate, 0.6% BSA) [24]. Sperm motility was evaluated and sperm concentration determined using a hemocytometer. The suspension was diluted to 25 x 106 sperm/ml with fert-TALP (114 mM NaCl, 3.2 mM KCl, 2.0 mM CaCl2, 0.5 mM MgCl26H2O, 25 mM NaHCO3, 0.34 mM NaH2PO4H2O, 10 mM sodium lactate, 100 IU/ml penicillin, 0.2 mM pyruvate, 0.6% BSA) [24]. Oocytes were removed from maturation medium, rinsed in fert-TALP, and placed in multiwell culture dishes. A 20-µl aliquot of diluted sperm (for a total of 500 000 sperm per well or 1 x 106 sperm/ml) and 20 µl of heparin (Sigma; 50 µg/ml in fert-TALP) were added to each fertilization well containing up to 50 oocytes in 0.5 ml of fert-TALP. Frozen oocytes were inseminated approximately 20 min postthaw. They were rinsed and placed in culture medium (TCM 199+10% Fetal Clone) 18 h postinsemination and fixed between 62 and 66 h postinsemination. Oocytes were stained with Hoechst stain and examined using fluorescence microscopy for fertilization and cleavage.
Oocyte Fixation and Staining
Prior to fixation, cumulus cells were removed from oocytes by high-speed vortexing in about 50 µl of fert-TALP (2 min for unfrozen oocytes and 1 min for frozen-thawed oocytes). Oocytes that were to be stained for chromatin only were fixed immediately in 3% formalin in PBS containing 0.1% sodium azide and 0.3% BSA (PBS-azide). Oocytes were then stored in fixative diluted with PBS-azide until examination. Oocytes for chromatin, tubulin, and actin staining were fixed and extracted in a modified microtubule-stabilizing buffer (0.1 M Pipes, 5 mM MgCl2, 2.5 mM EGTA, 0.01% aprotinin, 1 mM dithiothreitol, 50% D2O, 0.1% Triton X-100, 3% formalin) [26] for 1 h at 37°C. The time between removal from the incubator to placement in fixative varied from 4 to 7 min. After fixation, oocytes were rinsed briefly and stored at 4°C in PBS-azide supplemented with 0.76% glycine until examination.
All incubations and rinses were done at about 25°C. Fixed oocytes to be stained for chromatin only were rinsed briefly in Hanks' Balanced Salt Solution containing 0.05% saponin (washing solution, WS) and 0.5% BSA and were incubated in Hoechst 33258 (Sigma; 10 µg/ml) for 2 min. Oocytes to be stained for chromatin, tubulin, and actin were incubated in PBS-azide supplemented with 3% skim milk for 20 min. Oocytes were then rinsed four times in WS containing 0.1% BSA [27] and incubated in prediluted mouse 
-anti-tubulin (Zymed, South San Francisco, CA) for 1 h. Oocytes were then rinsed in WS+0.1% BSA three times, 20 min each, and placed again in PBS-azide with 3% skim milk for 20 min. After rinsing four times in WS+0.1% BSA, oocytes were incubated in fluoresceinated anti-mouse Ig whole antibody (from sheep; Amersham, Arlington Heights, IL) diluted 1:40 in WS+0.9% BSA for 4 h; they were then rinsed again three times, 20 min each, in WS+0.1% BSA. Oocytes were then incubated in rhodamine phalloidin (Molecular Probes, Eugene, OR) diluted to 0.5 U/ml in WS+0.5% BSA for 20 min. After this incubation, oocytes were rinsed five times, 2 min each, in WS+0.1% BSA and then incubated in Hoechst 33258 for 2 min. Oocytes were rinsed briefly in WS+0.5% BSA before being mounted on a slide in PBS:glycerol (1:1). Coverslips were sealed on slides using clear nail polish, and slides were stored at 4°C for 1–2 days until microscopic examination.
Microscopic Examination
Oocytes were examined routinely using a Zeiss (Carl Zeiss, Thornwood, NY) microscope with both epifluorescence and phase-contrast optics at a total magnification of x500. Four components of each oocyte were examined and categorized: chromosome arrangement, microtubule distribution, filamentous actin distribution, and the appearance of the cortical actin band (see Table 1). Photomicrographs were taken at a magnification of x400 using a Nikon (Garden City, NY) Diaphot 300 inverted microscope equipped with a UFX-DX photomicrographic attachment using Kodak (Eastman Kodak, Rochester, NY) Tmax 400 film.
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Experiment 1: Oocyte Cooling and EG Exposure
In a preliminary experiment, two periods of oocyte nuclear maturation were evaluated to establish the earliest time when oocytes were consistently in metaphase II. Based on these results (see Table 2), oocytes in subsequent experiments were matured 24 h before use.
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Oocytes were randomly divided into 4 treatment groups after 24 h of maturation. One group was maintained at 39°C in fert-TALP throughout the treatment period. A second group was placed in a Petri dish containing DPBS+0.4% BSA (DPBS-BSA) at about 25°C and within 10 min were loaded into 0.25-ml plastic straws (20–40 oocytes per straw). Straws then were placed directly into a BioCool programmable methanol freezer (FTS Systems, Stone Ridge, NY) at -9°C for 12 min, and finally rewarmed in a water bath at about 25°C. Straws were emptied and oocytes were recovered and transferred into fert-TALP.
A third group of oocytes was equilibrated in 1.5 M EG in DPBS-BSA for 10 min. EG was then removed by transferring oocytes to 0.75 M EG+0.45 M galactose for 5 min, 0.25 M galactose for 5 min, and finally DPBS-BSA for 5 min before transfer to fert-TALP. All transfers were made at about 25°C.
Oocytes in the final treatment group were equilibrated with EG as above, loaded into straws, cooled to -9°C for 12 min, and recovered from straws; EG was then removed as described above. After all treatments, oocytes were incubated at 39°C for 20 min or 1 or 3 h and then vortexed and transferred to fixative at about 25°C.
Experiment 2: Freezing and Thawing Oocytes
In the second experiment, oocytes in 1.5 M EG were loaded into straws (30–50 per straw) and equilibrated at -9°C as before for 2 min. Seeding was induced by touching individual straws with forceps cooled in liquid nitrogen. After seeding, straws remained at -9°C for 10 min and then were cooled to -40°C at 0.3°C/min [2] before transfer to liquid nitrogen. Oocytes were stored in liquid nitrogen for 4 days to 1 mo before thawing.
Straws were removed from liquid nitrogen, held in air for 10 sec, then placed in a water bath at 25°C for 3 min. After EG removal as in experiment 1, oocytes were rinsed once in fert-TALP and placed in a Nunclon 4-well multidish (50 oocytes per well) containing 0.5 ml of fert-TALP. Concurrently, unfrozen control oocytes were removed from maturation medium, rinsed, and transferred to separate wells. Oocytes were incubated and fixed as in experiment 1.
Statistical Analysis
Experiments were replicated completely 4 times (the actin band was evaluated in 3 of 4 replicates), and data from all replicates was pooled for statistical analysis (40–80 oocytes per treatment). Oocytes were assigned a quality score of 1, 2, or 3 corresponding to a subjective description of normal, abnormal, or missing for each cytological component (see Table 1); ANOVA was performed using General Linear Models/ANOVA procedures in MINITAB (Minitab, Inc., State College, PA) to determine significance of the main effects of cooling, EG exposure, and time. Oocytes in the abnormal and missing categories were pooled, because of low numbers in the missing category, and compared with the normal category to determine differences between treatment groups using chi-square analysis on pair-wise combinations. Differences between maturational status and cleavage rate were compared using chi-square analysis of data pooled from 2 complete replicates.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Filamentous actin was examined separately for distribution throughout the oocyte and for the fluorescence intensity and continuity of actin immediately beneath the oolemma, referred to as the actin band. Actin in most untreated oocytes appeared homogeneously distributed with a distinctively more intense cortical actin band (Fig. 7). Oocytes with this appearance or with a slightly discontinuous or punctate actin staining pattern were classified as having a normal microfilament distribution and accounted for 62–88% of untreated oocytes (Figs. 8 and 9). The cortical actin band had a uniform width and brightness in > 80% of untreated oocytes (Figs. 7, 10, and 11). Oocytes with this appearance of the actin band or with slight variation in fluorescence intensity of the actin band were classified as normal.
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After cooling and rewarming, chromosome configuration remained normal for up to 1 h at 39°C, but abnormalities increased after 3 h of incubation (Fig. 3, 33% versus 84% for untreated oocytes). Cooling and rewarming affected microtubules in a time-dependent manner as well, with 60% of oocytes containing normal spindles 20 min after rewarming, 42% after 1 h, and only 28% after 3 h (Fig. 5). Actin distribution was not affected immediately by cooling. After rewarming, 92% of oocytes contained a normal distribution of microfilaments. This value decreased to 35% after 1 h and 28% after 3 h (Fig. 8). The actin band appeared to be disrupted by cooling initially, but the percentage of oocytes with a normal actin band actually increased from 41% to 59% after 3 h at 39°C (Fig. 10).
Exposure to EG did not affect chromosome arrangement initially, although a lower percentage of oocytes with normal chromosome configuration was seen after 3 h (Fig. 3, 56% versus 84% of untreated oocytes). The percentage of oocytes with normal spindles was not significantly different from that for untreated oocytes after 20 min, but it decreased after 1 and 3 h (Fig. 5). EG exposure decreased the percentage of oocytes with normal microfilament distribution initially, but this percentage returned to the levels of untreated oocytes after 1 and 3 h (Fig. 8), and normal structure of the actin band also appeared to be restored with time (Fig. 10).
Cooling oocytes loaded with EG was more disruptive to chromosome configuration than cooling alone. The percentage of oocytes with normal chromosome arrangement was not significantly different from that for untreated oocytes 20 min after treatment but decreased over time, from 71% at 20 min to 26% at 3 h (Fig. 3). Fewer oocytes contained normal chromosome configuration by 1 h of incubation than untreated, cooled, or loaded oocytes. Only 41% of oocytes contained a normal spindle 20 min after treatment, and this value decreased even further to 12.5% after 3 h (Fig. 5). Oocytes loaded with EG and cooled seemed to retain normal microfilament distribution better than oocytes cooled without EG, but there were still significantly fewer oocytes with normally distributed microfilaments than in untreated oocytes after 1 h and 3 h of incubation (Fig. 8). A smaller percentage of oocytes containing normal actin bands was observed in the combined treatment as well as after 1 and 3 h of incubation (58–63% versus 80–87% in untreated oocytes, Fig. 10).
Freezing had a significant deleterious effect on all cellular components examined. Fewer frozen-thawed oocytes in each treatment group had a normal chromosome arrangement than unfrozen oocytes. At 20 min postthaw, 64% of frozen oocytes contained normal chromosome configuration, but this value decreased to 30% after 3 h (Fig. 4). Over 70% of frozen oocytes had missing spindles by 20 min postthaw, and only 12% had normal spindles (Fig. 6).
Freezing also had a dramatic effect on cytoskeletal actin (microfilaments). The percentage of oocytes with normal microfilament distribution was significantly lower than that of unfrozen oocytes for each treatment group. After 3 h, 61% of oocytes had disorganized, faint staining of actin, compared to 6% for unfrozen oocytes (Fig. 9). Areas of fluorescence were still visible in the frozen oocytes, but the distribution was discontinuous. The actin band was also affected by freezing, with only 23–28% of frozen oocytes containing normal band structure (Fig. 11), a value significantly lower than that for controls (83–89%).
Freezing oocytes significantly decreased the rate of cleavage to 2 cells or more at 62–66 h postinsemination, from 53% in control oocytes to 8% for frozen oocytes (Table 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Microtubules were confined to the meiotic spindle in most untreated oocytes, but cooling resulted in a progressive disorganization of the spindle. As with human oocytes [13], the spindle of chilled bovine oocytes is not completely restored upon rewarming [18]. This appears to be due to a lack of pericentriolar material (PCM) available for nucleating microtubule formation in the oocytes of these species because the spindle of chilled mouse oocytes, which have a relative abundance of PCM, reassembles upon rewarming [29].
Cryoprotectants (DMSO and PrOH) have been shown previously to cause changes in microtubule organization in several species, including the mouse [10, 29], rabbit [16], and human [12]. Exposure of bovine oocytes to EG increased abnormal spindle formation. EG may affect the dynamic assembly and disassembly of microtubules, as has been shown for DMSO [16]. However, in this study, the effects of EG exposure cannot be separated entirely from those of cooling to room temperature (the temperature at which EG was loaded).
EG exposure combined with cooling disrupted microtubule organization more than cooling alone. Vincent and Johnson [29] concluded that although exposure to DMSO or cooling alone is damaging to oocytes, the combined treatment stabilizes the spindle against cooling-induced depolymerization because less CPA enters the cell when loaded at a lower temperature. In this study, however, effects of cooling and CPA exposure were cumulative, possibly due to increased permeability of CPA at room temperature.
Chilling and CPA exposure consistently result in variable displacement of chromosomes from the metaphase plate associated with altered spindle morphology [7, 9, 13, 16, 18]. Gook et al. [30] suggested that chromosomes of human oocytes are anchored by associated kinetochores and do not move about freely within the cytoplasm even after cryopreservation. However, spindle disruption of bovine oocytes caused by chilling or CPA treatment preceded chromosome disorganization by several hours.
Actin staining revealed craters and apparent holes in the plasma membrane of chilled bovine oocytes. Cooling does not affect the microfilaments of mouse oocytes as drastically as it does microtubules, although some alterations in organization may be seen along with distortions of the oocyte [9, 29]. Normal microfilament distribution in cooled and rewarmed bovine oocytes decreased in general over time while the appearance of normal actin bands increased, reflecting some recovery of continuity in the actin band but loss of sphericity. In fact, fluorescence in noncortical regions of the oocyte may originate secondarily to fluorescence of the actin band due to lack of confocality in the epifluorescent images. Williams et al. [17] observed that filamentous actin appeared almost exclusively in the cortical band of bovine oocytes when examined using confocal microscopy.
Because cortical actin is closely associated with the plasma membrane, alterations in microfilament distribution after cooling may be a result of damage to the plasma membrane rather than a direct result of cooling. The phase behavior and physical properties of membrane lipids are sensitive to acute changes in temperature [31]. Didion et al. [32] observed that cooling below 15°C caused damage to the plasma membrane of pig oocytes. Little else has been reported on the effects of cooling on the plasma membrane of oocytes, however. We have observed that cryopreservation reduces the osmotic responsiveness of in vitro-matured (IVM) bovine oocytes from 83–92% to only 36–47% (unpublished results), indicating that the plasma membrane has been compromised and that cytological effects may be secondary to altered membrane permeability.
Exposure to EG in this study did not lead to substantial disruption in microfilament organization, though previous studies have shown varying effects of CPAs on rabbit and mouse oocytes [8, 16, 19]. In studies in which microfilament disorganization was observed, oocytes were fixed without removal of the CPA. Thus it is possible that EG does destabilize microfilaments in bovine oocytes and that this effect is reversed after CPA removal. Microfilament organization in bovine oocytes loaded and unloaded with EG or PrOH was similar to that in untreated oocytes [17]. Oocytes loaded with DMSO were characterized by a punctuated appearance after actin staining, and after unloading contained discontinuous actin bands and craters in the actin pattern. These results support observations in the present study indicating only a minor effect of EG on actin distribution. In contrast to the effects on microtubules, normal microfilament distribution was more often observed in oocytes that were loaded with EG before cooling than in those cooled without EG, indicating a protective effect of EG during cooling.
Disruption of the cytoskeleton may be intrinsic to changes in shape and shrinkage that accompany cryopreservation procedures, which in turn may lead to premature release of cortical granules and zona hardening [29, 33] by allowing cortical granules to localize subjacent to the plasma membrane. Rewarming may then lead to membrane fusion and release of enzymes. Thus, even when normal microfilament distribution of oocytes is restored after cooling or CPA exposure, irreversible alterations of other cellular components may already have occurred. Release of cortical granule enzymes and premature zona hardening may either block fertilization completely or incompletely block polyspermy, both cases resulting in a decreased cleavage rate after insemination.
Freezing causes drastic changes in organization of the meiotic spindle and chromosomes in mouse [34], human [20], and rabbit oocytes [16]. In this study, freezing bovine oocytes led to the complete disappearance of spindles in over 70% of oocytes. Normal spindle structure was not recovered even after 3 h of incubation. Normal chromosome configuration decreased to 30% within 3 h. George and Johnson [22] reported that the percentage of frozen-thawed mouse oocytes with normal spindles and chromosome arrangement was not different from that of controls immediately postthaw but was lower than that of controls 3 h later.
Freezing also causes changes in the organization of cytoskeletal actin in rabbit and mouse oocytes [16, 22], although these changes were not dramatic and were often reversible upon thawing. The present study shows a dramatic change in distribution of actin in bovine oocytes after freezing-thawing. Many oocytes with disrupted microfilament organization also had a discontinuous or lysed plasma membrane as evidenced by observations with phase-contrast microscopy. Because of the association of microfilaments with other structures, it is possible that their disruption is the result of damage to another cellular component such as the plasma membrane or mitochondria. After freezing-thawing pig oocytes, Didion et al. [32] observed that none survived the process as evidenced by use of vital stains that depend on membrane integrity. Disruption of the plasma membrane and mitochondria also have been observed in frozen-thawed human [20] and bovine oocytes [4, 23]. Increased permeability to calcium or loss of mitochondrial function would be expected to alter the dynamic assembly and disassembly of both microtubules and microfilaments. The osmotic stress, solute effects, or intracellular ice formation intrinsic to the freezing process may irreversibly change the structure of the plasma membrane or cytoskeleton so that it can no longer adjust to changing conditions, including those changes brought about by sperm penetration. Not all oocytes penetrated by sperm in this study developed further, and polyspermy did not account completely for arrested development. Eroglu et al. [35] reported an inhibition of pronuclear formation in frozen-thawed mouse oocytes that requires microfilaments [29], suggesting that cytoskeletal alteration may be involved.
The limitations of using oocyte morphology as a basis for postthaw survival are apparent from this study. When oocytes are examined for survival without cumulus removal, high values may be obtained because of a normal appearance [32]. When cumulus cells are removed, oocytes can be graded more objectively, but estimates of survival immediately postthaw remain an inaccurate indicator of survival as assessed by further development [5]. Evaluating several intracellular components in this study provided a more accurate assessment of oocyte survival. In addition, evaluation of cytological effects after posttreatment incubation suggested the occurrence of latent damage that was not evident immediately after exposure. Less than 30% of oocytes were normal postthaw when chromosomes, microtubules, or microfilaments were observed—a value more closely correlated with subsequent fertilization and cleavage rates than morphological appearance. Cytological effects of cryopreservation procedures may be exacerbated by or even secondary to plasma membrane damage, which may not be readily visible. Under conditions used in this study, 65% of oocytes were classified as morphologically normal postthaw but < 50% were osmotically responsive to 1.0 M sucrose (unpublished results), suggesting that the oolemma was compromised even in oocytes with a normal morphological appearance. These observations should be considered when reporting survival rates.
Because of the damage incurred during both prefreezing manipulations and the freezing process itself, it is increasingly apparent that minor alteration of conventional freezing parameters is unlikely to improve results for bovine oocytes [36]. Novel approaches to cryopreservation are needed to improve survival and development rates postthaw. Martino et al. [5] exposed bovine oocytes to high concentrations of EG plus sucrose for less than 30 sec on electron microscope grids, then plunged them into liquid nitrogen. After thawing, cleavage rates of 25–40% were obtained compared to 3% for oocytes vitrified in plastic straws. Cleavage rates of 50% were achieved by Vajta et al. [37] using an open pulled straw vitrification technique. In these procedures, damage due to prolonged exposure to low temperature or CPA may be prevented because the cooling rate "outpaces" temperature effects on the oolemma and other cytological features [5, 37]. This may be the basis for improvements in bovine oocyte cryopreservation using ultrarapid and related procedures [5, 37–39]. Even with these recent improvements, however, the rate of cleavage and blastocyst formation still remains well below control levels. A better understanding of how these approaches overcome cytological damage associated with conventional procedures, in addition to the effects of maturation stage [40] and membrane permeability characteristics [41, 42], should provide a fundamental basis for the improvement of developmental potential of cryopreserved oocytes from cattle and other mammalian species.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: John E. Parks, 201 Morrison Hall, Cornell University, Ithaca, NY 14850. FAX: 607 255 9829; jep5{at}cornell.edu
Accepted: February 23, 1999.
Received: September 16, 1998.
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REFERENCES |
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