Vitrification of mouse germinal vesicle oocytes: effect of treatment temperature and egg yolk on chromatin and spindle normality and cumulus integrity

E.F. Isachenko1 and P.L. Nayudu

Department of Reproductive Biology, German Primate Center, Kellnerweg 4, Göttingen, 37077, Germany


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The success rates for cryopreservation of immature oocytes from several species including human remain low, in contrast to major improvements with mature oocytes. In this study, a new approach has been developed using a short exposure ultra-rapid freezing protocol, examining the effect of temperature and egg yolk (two factors which may be expected to influence membrane flexibility) on the cryostability of immature mouse oocytes and cumulus complexes. These two factors were tested in various patterns for their cryoprotective effect using ethylene glycol as the principal cryoprotectant. The results showed that 37°C pre- and post-freeze exposure significantly improved both survival and normal spindle configuration after in-vitro maturation. Egg yolk was found to produce further beneficial effects on both the oocyte and cumulus cell integrity, with the best effects being obtained at 37°C with inclusion of egg yolk both before and after the freezing. This protocol produced >80% normal survival post-thaw with intact and attached cumulus complex, 84% maturation rate and 45% normal metaphase configuration. In summary, a unique combination of high survival and meiotic normality together with good preservation of the attached cumulus cell mass has been achieved using a simple new vitrification procedure.

Key words: chromatin/germinal vesicle oocytes/mouse/rapid freezing/spindle


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although cryopreservation of human embryos is both highly successful and widely used, it has created many ethical and legal problems (Koninckx and Schotsmann, 1996) and is now prohibited in some countries. A viable alternative may be the cryopreservation of oocytes, either mature or immature. To date only mature human oocytes have been successfully cryopreserved (Chen, 1986Go, 1988Go; Van Uem et al., 1987Go; Gook et al., 1993Go, 1994Go; Baka et al., 1995Go) using a propanediol slow freezing method similar to that used for embryos. The recent successful pregnancies using this method (Porcu et al., 1997Go; Polak de Fried et al., 1998Go) have provided the first confirmation of oocyte functionality.

In contrast, attempts to cryopreserve immature human oocytes using the same method have produced only limited success (Son et al., 1996Go) with extensive abnormalities of the metaphase spindle being reported (Park et al., 1997Go). The development of an effective method of cryopreservation for immature oocytes, with their attached cumulus cells, would offer an alternative to the storage of mature gametes or embryos. Immature oocytes could be stored immediately after collection and only matured and fertilized when required. This would be expected to result in a more convenient and simple routine for both the surgeon and the laboratory, with a further reduction in time and financial investment if rapid freezing were employed. Mammalian oocytes of all species tested, in particular immature ones, appear to be more sensitive than zygotes or embryos to the procedures involved with cooling or cryopreservation (Van Uem et al., 1987Go; Sathananthan et al., 1988Go; Parks and Ruffing, 1992Go). The temperature sensitivity of oocytes is likely to involve a number of different aspects of structure (Vincent and Johnson, 1992Go; Younis et al., 1996Go). Even without freezing, cooling is known to cause depolymerization of microtubules and disappearance of microtubule-organizing centres (Gulyas, 1975Go; Moor and Crosby, 1985Go; Pickering and Johnson, 1987Go; Didion et al., 1990Go).

One simple but important approach to reducing cellular damage may be to maintain physiological temperature during cryoprotectant exposure, in order to avoid shock to the oocyte. This would also be expected to increase membrane fluidity, thereby facilitating cryoprotectant entry (Isachenko et al., 1998Go), and secondarily increasing cytoskeletal flexibility (Hunter et al., 1990Go). However, higher exposure temperatures are associated with increased toxicity of permeable cryoprotectants (Aigner et al., 1992Go), so low toxicity permeable cryoprotectants such as ethylene glycol (Smyth et al., 1950Go; Bautista and Kanagawa, 1998Go) are necessary. Further, the well known membrane stabilizing effects of egg yolk, previously applied mainly to sperm cryopreservation [to our knowledge the single exception was reported for porcine embryo vitrification (Fujino et al., 1993Go)] may also be used to advantage. Certain lipidic elements of egg yolk (Kuksis, 1992Go) may combine with cell membranes, altering their molecular composition and maintaining their fluidity (Holt et al., 1992Go; Ostashko, 1995Go; Gamzu et al., 1997Go).

In the present study mouse oocytes have been used as an easily available source of low lipid oocytes (more similar to human than those of domestic species such as bovine and porcine) for the initial method development. We have investigated the effects of temperature and egg yolk on the cryostability of immature mouse oocytes after vitrification in ethylene glycol. OCC were evaluated by post-thaw survival, capacity for maturation, normality of the meiotic apparatus and integrity of the cumulus mass. Fertilizing potential has not been tested in this series of experiments.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals and oocyte recovery
F1 virgin female mice (C57 BL/6JxCBA/Ca), 21–23 days old (Harlan Winkelmann GmbH, Borchen, Germany) were used in this study. The experiments were conducted according to German animal protection laws. Mice were injected with 5 IU of pregnant mare serum gonadotrophin (PMSG, Intergonan; Vemi Veterinär, Kempen, Germany) and killed after 40 h. Six to eight ovaries (160–200 oocytes) were collected in Leibovitz L-15 medium (Gibco, Berlin, Germany) with 5% heat-inactivated fetal bovine serum (FBS). Oocyte–cumulus complexes (OCC) were recovered by puncture of antral follicles. In all experiments, only OCC (n = 1105) with compact and dense cumulus cell layers were used and partly or completely naked oocytes were discarded. From this pool the oocytes were randomly allocated to treatment groups.

Vitrification
Except where otherwise indicated, chemicals were supplied from Sigma Chemical Co. (Deisendorf, Germany).

OCC were divided into 20 treatment and one control groups, described in Table IGo. The experiment was based on a modification of the technique described by Miyake et al. (1993) which employs ethylene glycol and sucrose, 25°C equilibration temperature and ultra-rapid freezing. The new aspects investigated here were a higher temperature of exposure (37°C) compared with 25°C and the effects of egg yolk in different patterns of exposure. The vitrification solution was prepared in phosphate-buffered saline (PBS) medium modified by Wood et al. (1987) supplemented with 5% FBS (holding medium). Egg yolk solution was prepared from eggs laid no more than 12 h previously. Fresh egg yolk immediately after recovery was mixed with holding medium 1:2 and centrifuged for 50 min at 10000 r.p.m. at 15°C. Only the supernatant was used for the protocols described in Table IGo. Straws were partially filled with a column of 50 µl of cryoprotectant medium containing oocytes (5–7 per straw), then sealed with a plastic plug (Mini-tüb, Landshut, Germany) and plunged immediately into liquid nitrogen. The oocytes were stored in liquid nitrogen for at least 24 h before being thawed. They were thawed by holding the straw for 10 s at room temperature in air and then subsequently transferred to a water bath and gently agitated at 37°C. After dilution of cryoprotectant OCC were transferred into maturation medium. The cryoprotectant was removed immediately after thawing by expulsion of the oocytes together with the vitrification solution into 1 ml of dilution medium for 5 min. The oocytes were washed three times for 5 min in holding medium. After this, the OCC were transferred into maturation medium. A random sample of oocytes was evaluated for maturation status at this stage and all were in GV stage.


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Table I. Experimental design: Effect of the temperature and egg yolk applied in different exposure patterns
 
Maturation of oocyte–cumulus complexes (OCC)
Oocytes exposed to cryoprotectants without freezing (non-frozen control group) as well as those of experimental groups after dilution of cryoprotectants, were exposed to in-vitro maturation conditions in order to evaluate their capacity to mature normally. The maturation procedure was a modification of Eppig (1991), as reported by Gilchrist et al. (1995). Groups of 5–7 OCC were transferred to individual 50 µl droplets of Waymouth's Medium MB 752/1 (Gibco, Berlin, Germany) supplemented with 1 µg/ml human FSH [NIDDK-hFSH-B1 (AFP-8792B)], 10 µg/ml human LH [NIDDK-hLH-B-1 (AFP-8792B)], 20% FBS, 0.5 mM Na pyruvate, 10 mM Na lactate, 4 mM hypotaurine, 1 mM glutamine, 75 mg penicillin G-K salts and 50 mg streptomycin sulphate under non-washed liquid paraffin oil (BDH, Wesel, Germany) in 4-well culture dishes (Nunclon, Roskilda, Denmark). Oocytes were cultured for 17 ± 1 h in a humidified atmosphere of 5% CO2 in air.

Evaluation of oocytes
OCC were classified after maturation as surviving the exposure in the cryoprotectant mixture and/or the vitrification procedures if they displayed a clear bright and homogeneous cytoplasm and an intact zona pellucida, and virtually no space between the zona pellucida and the cytoplasm. Integrity of the cumulus cell layers after vitrification was assessed by pipetting the OCC up and down to determine whether the cumulus cells were firmly attached and after culture by expansion and attachment of cumulus cells.

After culture, the oocytes were classified based on the presence or absence of a polar body. Among those oocytes which had extruded a polar body during maturation, normal metaphase (MII) was defined as the oocytes with both normal MII chromosomes and with a barrel-shaped spindle and a tubulin and chromatin-containing polar body, as evaluated by fluorescent methods. For the evaluation of normality of the spindle and chromatin we have used very strict criteria, within the limits of our imaging capabilities. The metaphase figure was considered normal only when it satisfied all criteria of normal arrangement of chromosomes and spindle fibres. Minor variations such as displaced spindle fibres, chromosomes out of alignment or small spindles, were classified as abnormal.

Chromatin and spindle visualization
Chromatin and spindle configuration were assessed using the fluorescent methods described in Gilchrist et al. (1995). The procedure was as follows: oocytes after removing of cumulus-cell masses with 0.5 mg/ml hyaluronidase (for 1–3 min), were fixed in prewarmed (37°C) 2% paraformaldehyde (Merck, Darmstadt, Germany) in Dulbecco's phosphate-buffered saline (DPBS) with 0.04% Triton X-100 (Serva, Heidelberg, Germany) for 1 h, and stored overnight at 4°C in DPBS with 0.3% (w/v) bovine serum albumin (BSA, Fraction V) until staining and labeling. For spindle visualization, fixed oocytes were incubated in mouse monoclonal anti-{alpha}-tubulin (1/2500; ICN, Meckenheim, Germany) in DPBS with 0.3% (w/v) BSA for 45 min at 37°C. Oocytes were then washed in DPBS with 0.3% (w/v) BSA and 1 µl/ml Tween-20 (Merck, Darmstadt, Germany) for 90 min at 37°C, and then stained with fluorescein isothiocyanate (FITC) conjugated to goat anti-mouse F(ab')2 (1/100; ICN, Meckenheim, Germany) together with 10 µg/ml Hoechst 33258 in DPBS + 0.3% (w/v) BSA for 45min at 37°C in the dark. After the second wash (90 min at 37°C), oocytes were impregnated with Mowiol 4-88 mounting medium (Hoechst, Wiesbaden, Germany). The meiotic status of the stained oocytes was assessed with an Axiovert microscope under epifluorescence illumination with FITC excitation at 490 nm and Hoechst at 365 nm. Samples were photographed on Kodak Ektachrome 400 ASA colour positive film (Eastman Kodak, Rochester, NY, USA).

Statistical analysis
Data were analysed using an analysis of variance computer program. Differences were considered significant at P < 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effect of temperature and egg yolk on oocyte maturation after treatment with cryoprotectant solutions without freezing.
The effects of the various protocols applied without freezing (A – E0–1) on morphological survival, maturation rate and proportion of oocytes with normal spindles and chromosomes after dilution of cryoprotectant are shown in Table IIGo (ranging from 74–79%). In no treatment did the exposure to any of the cryoprotectant mixtures cause a reduction in oocyte survival and no effect was noted on the cumulus cells, indicating a lack of major toxic effects. There was, however, a significant reduction in maturation rate for the treatment group not containing egg yolk (E0,1) below that observed for the untreated group, with the higher temperature producing no improvement. For those oocytes which matured, there was no reduction in the proportion of normal meiotic figures in any of the treatment groups compared with the untreated control group.


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Table II. Effect of temperature and presence of egg yolk on oocyte maturation after exposure to and dilution from cryoprotectant solution at 25°C or 37°C without freezing
 
At 25°C, the inclusion of egg yolk in either cryoprotectant or dilution media produced no improvement in maturation rate (C0 and D0) compared with exposure to ethylene glycol alone. But when egg yolk was included in both media (A0 and B0), a 15–20% improvement in maturation rate was produced, with both application patterns being equally effective. At 37°C, an increase in maturation rate of 11 and 15% respectively produced by inclusion of egg yolk in either cryoprotectant or dilution media (D1 and C1), indicated an increased efficacy at the higher temperature. When egg yolk was present in both media at 37°C (A1 and B1), a >20% increase in maturation rate over the exposure at the same temperature without egg yolk was observed (~10% higher than for the same treatment at 25°C). The best two groups (A1 and B1) had maturation rates very close to that of the untreated control group. Therefore, the cumulative effect of temperature and double treatment with egg yolk virtually eliminated the negative effect of exposure to ethylene glycol and sucrose on maturation rate.

Effect of temperature and egg yolk on post-thaw OCC
Survival, maturation and meiotic normality
The effects of the various protocols applied with freezing (A – E2–3) on post-thaw morphological survival, maturation rate and proportion oocytes with normal spindle and chromosomes are shown in Table IIIGo. For all protocols, freezing produced a drop in survival. Exposure to ethylene glycol at 25°C without egg yolk resulted in a reduction in survival to ~50%, while exposure at 37°C provided better protection (~70% survival). At 25°C, the inclusion of egg yolk in either freezing or dilution media (C2 and D2) did not produce better survival compared with ethylene glycol alone, while exposure at 37°C for the same protocols (C3 and D3) produced only a slight improvement above the comparable ethylene glycol group without egg yolk. The highest survival rates occurred at 37°C with the double exposure to egg yolk (A3 and B3), although at 25°C (A2 and B2) the double exposure groups also produced an improved survival over single and no exposure. In overview, 37°C provided better survival, and this was augmented only by double exposure to egg yolk, with the best treatment producing 83% survival rates.


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Table III. Effect of temperature and egg yolk exposure on maturation of vitrified GV mouse oocytes
 
The rate of in-vitro maturation was influenced by the presence of egg yolk but not by temperature, as with the non-frozen treatment groups above. Exposure to egg yolk before freezing provided more effective protection and produced a better maturation rate, perhaps not unexpectedly, than exposure only after thawing. The double exposure to egg yolk produced the best effect with a maturation rate not significantly different from the untreated controls but temperature did affect normality. The proportion of oocytes with normal metaphase figures produced at 37°C was significantly better than with 25°C for all groups containing egg yolk. Without egg yolk, the effect was much less marked and the proportion of normal figures significantly lower. Figure 1AGo shows an example of normal chromatin and spindle configuration after freezing and in-vitro maturation after. Examples of the common abnormal forms are illustrated in Figure 1B–D.Go



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Figure 1. Examples of normal and abnormal chromatin and spindle configurations after freezing and subsequent in-vitro maturation. (A) Normal chromatin and spindle configuration after freezing and maturation. (B) Small spindle and chromatin which is distributed out of the spindle's limit. (C) Major disorganization of meiotic apparatus. (D) Minor disorganization of chromatin within spindle. For all micrographs bar = 7.4 µm.

 
OCC integrity
The integrity of the cumulus mass after freezing has been compared among the various egg yolk exposure protocols and with ethylene glycol without egg yolk and the untreated control group (Table. IVGo). Temperature had no major effect on the attachment of the cumulus to the oocyte; however, the exposure to egg yolk (regardless of pattern) produced a significant improvement in cumulus integrity with a high proportion of oocytes having tightly attached cumulus cells, as seen in Figure 2AGo. In contrast, without egg yolk a proportion of oocytes had non-attached or absent cumulus (Figure 2BGo). Further, viability of these cells was compared after in-vitro maturation by their capacity to attach and spread on the plate. OCC treated with egg yolk attached and spread more vigorously on the plate (Figure 2CGo), than those not treated with egg yolk (Figure 2DGo).


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Table IV. Integrity of cumulus-cell layers after thawing and in-vitro maturation (IVM) according to temperature of equilibration and dilution of cryoprotectant
 


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Figure 2. Effect of egg yolk on the cumulus cells. Immediately after thawing and pipetting: (A) with egg yolk treatment; (B) without egg yolk treatment. After in-vitro maturation: (C) with egg yolk treatment; (D) without egg yolk treatment. With egg yolk (A) a high proportion of cumulus complexes remained intact in contrast to those without egg yolk, where a high proportion of cumulus cells was loose or absent (B). After maturation with egg yolk, the cells plated and spread vigorously (C) in contrast to those not exposed to egg yolk (D). For all micrographs bar = 58 µm.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Human immature oocytes have not yet been successfully cryopreserved (Son et al., 1996Go; Park et al., 1997Go). Immature mouse oocytes have been cryopreserved using a vitrification cryoprotectant mixture (Van Blerkom, 1989), an ultra-rapid method (Van der Elst et al., 1992Go, 1993Go) as well as slow freezing methods (Schroeder et al., 1990Go; Candy et al., 1994Go; Karlsson, et al., 1996Go; Matson, et al., 1997Go; Cooper et al., 1998Go) employing dimethylsulphoxide (DMSO) or propanediol as cryoprotectants, but with limited success. Those studies which have compared the cryosensitivity of immature with mature mouse oocytes (Schroeder et al., 1990Go; Candy et al., 1994Go) have all indicated the greater cryosensitivity of immature oocytes and reported low embryo development rates.

In the present study, we have adopted a new approach for cryopreservation of immature oocytes, aimed at the protection of membrane integrity as a means to preserve internal cytostructure. An ultra-rapid freezing method was employed using a relatively high concentration of ethylene glycol as cryoprotectant (adapted from Miyake et al., 1993Go). This cryoprotective agent was chosen because it is known to be less toxic and more compatible with high survival rates in tests with mature mouse oocytes (Rayos et al., 1994Go; Hotamisligil et al., 1996Go) and bovine embryos (Niemann, 1991Go; Suzuki et al., 1993Go) than other commonly used cryoprotectants. This is particularly important with the high exposure temperature which has been tested in this study. Further, ethylene glycol is known to have permeability properties only slightly inferior to those of DMSO, a much more toxic cryoprotectant (Speth and Wunderlich, 1973Go; Vincent et al., 1990Go). The two variables tested in this study, temperature of cryoprotectant exposure and dilution (25°C or 37°C) and the effect of egg yolk, were chosen as approaches likely to reduce the stress imposed on the oocyte during exposure to cryoprotectant and cryopreservation.

Temperature during cryoprotectant exposure and dilution is an important factor because it is known that oocytes can be damaged simply by cooling. This appears to be related to effects on various elements of the cytoskeleton (Parks and Ruffing, 1992Go; Vincent and Johnson, 1992Go; Williams et al., 1992Go; Ruffing et al., 1993Go). Since exposure to cryoprotectants is also damaging (the degree depending on the cryoprotectant involved), the normal approach has been to expose the cells at a cool temperature (Schalkoff et al., 1989Go; Depiesse et al., 1991Go; Fuku et al., 1992Go) which is unlikely to be optimal for oocytes and may have contributed to the poor results to date. However, when using temperatures of 25°C or above, the exposure time must be as short as possible and cryoprotectants of low toxicity must be employed to minimize damage from the cryoprotectant, as shown by the studies of Hotamisligil et al. (1996) on the effects of ethylene glycol on mature mouse oocytes.

The biophysical basis of cryodamage in oocytes is incompletely understood, but sufficient information exists on which to base the design of a new approach. Mammalian cells generally are not cold-adapted, and oocytes are particularly sensitive. Cooling would be expected to reduce membrane fluidity and although exposure of individual membrane constituents to extreme conditions is unlikely to affect physical structure or denature the components (Quinn, 1989Go), disturbance to the organization of the lipids is likely to damage, possibly irreversibly, the structure and function of the membrane. One of the more severe types of damage is lyotropic phase transition in the plasma membrane lipids, which is likely to occur during cooling (Singer and Nicolson, 1972Go; Didion et al., 1990Go) due to thermotropic phase-transition of polar lipids. The consequent lateral phase separation of membrane lipids and proteins (Quinn, 1989Go) and exclusion of the proteins from the bilayer tends to be irreversible after thawing (Speth and Wunderlich, 1973Go).

It is likely that the maintenance of temperature as close to physiological as possible until commencement of the freezing process would tend to reduce phase transition changes. This would in turn tend to reduce alterations to the interconnected cytoskeleton. The factors which are likely to contribute to the greater sensitivity of the immature oocyte compared to the mature are the specific structure of the zona pellucida, cytoskeleton, cytoplasmic membranes and their composition (Sathananthan, 1994Go; Zhong, et al., 1997Go) and the connections of the cumulus-cell projections and the oocyte cytoskeleton through their junctions with the oolemma (Allworth and Albertini, 1993Go). It is important that these connections remain intact for the completion of normal maturation in vitro (Buccione et al., 1987Go; Mattson and Albertini, 1988Go; Vanderhyden and Armstrong, 1989Go). However, where the cumulus cells have been mentioned in oocyte cryopreservation studies, it was noted that they were no longer connected to the oocyte after thawing (Cooper et al., 1998Go).

It would appear therefore, that the cytoplasmic structure is a more critical factor in cryodamage than risk of damage to the metaphase spindle of mature oocytes (Bernard and Fuller, 1996Go; Park et al., 1997Go), and it is the cytoskeletal damage, perhaps including the spindle organizers, that is the source of the spindle disorganization seen after post-thaw in-vitro maturation. In our study, we have seen that the severity of spindle and chromatin abnormalities occurring was much less extreme than that produced by DMSO at the same temperature (Vincent et al., 1990Go; E.F.Isachenko et al., unpublished results) with >90% of spindles from the protocol using egg yolk and 37°C temperature returning a barrel shape. This indicated therefore that the cytoskeletal damage was also significantly less with our method than with DMSO. Further, apparent discrepancies between reported high proportions of oocytes with barrel-shaped spindles after freezing and subsequent low embryo production (Van Blerkom, 1989; Cooper et al., 1998Go) suggest that less obvious defects of spindle/chromatin, which may not have been detected by low resolution methods, might also be expected to affect embryonic development negatively. Battaglia et al. (1996), using high resolution confocal microscopy to study the relationship between spindle defects and ageing, supported this supposition. We have therefore chosen to evaluate as abnormal, spindles with only minor defects such as barrel shaped with presence of fibres not attached at both ends and minor or major chromosomal displacement as well as disturbances in spindle symmetry and small size spindles.

Based on the above information regarding the interrelationships of the cytoskeleton and the cellular membrane, agents which protect the integrity of the cellular membrane during the cooling, freezing and thawing steps might also be expected to produce an improvement in the survival and normality of the oocytes after exposure to cryoprotectant and cooling. Egg yolk, a natural complex mixture of phospholipids and antioxidants, has been used for this purpose with spermatozoa for many years, but its use for oocyte cryopreservation to our knowledge has not been reported. Although it may not be desirable in future to use a natural and incompletely defined substance such as egg yolk with human oocytes, our observations of its benefits provide a starting point for future studies of defined lipidic mixtures.

The protective effect of egg yolk was augmented at the higher temperature of exposure possibly due to the greater flexibility of the cell structures at this temperature. How this protective effect functions is not entirely clear since egg yolk is such a complex mixture, but it may play a role in reducing the deleterious effects upon membrane structures of hyperosmotic salt solutions which occur during rapid cooling (Holt et al., 1992Go; Ostashko, 1995Go; Katkov et al., 1996Go). Some of the egg yolk components may also be incorporated into the membranes reducing their tendency to gel during cooling, as described Ostashko (1978, 1995) for sperm and erythrocytes. So the action may be a dual one, where the membrane is coated and isolated from direct contact with the cryoprotective agents and also maintains more fluidity and flexibility to a lower temperature, thereby reducing cytoskeletal disruption within the oocyte and cumulus cells. The fact that egg yolk exposure before freezing functioned equally well whether it was applied before or with the cryoprotectant indicated that its mechanism of action was independent.

Our study has shown that the two factors, increased temperature of exposure and egg yolk, effectively complemented each other. Even without freezing, protective benefits were noted with the nearly complete blocking of the drop in maturation rate induced by a short exposure to ethylene glycol without freezing. Further, cryoprotectant equilibration and dilution at 37°C was effective in improving survival after freezing even without egg yolk, but egg yolk appeared to work together with temperature and the combination significantly improved survival after freezing. The use of egg yolk at 37°C also improved the maturation rate and the proportion of normal metaphase figures, suggesting that it had a major effect on reducing internal cell damage. Finally, a very important factor is that this combination of egg yolk and higher temperature also preserved the integrity of the cumulus complex, perhaps by protecting the junctions of the cumulus-cell processes with the oolemma.


    Acknowledgments
 
E.F.I. was supported for part of this work by a BMBF cooperative grant with Ukraine (Project #UKR-027–96), and by a German Primate Center Fellowship. The authors wish to thank Ms Petra Kissei, Nicole Nüsse and Su Bete for their technical assistance, Dr Mauvis Gore for her help in statistical analysis, and Prof. J.K.Hodges for his continued interest in and support of this project.


    Notes
 
1 To whom correspondence should be addressed Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on July 13, 1998; accepted on October 22, 1998.