Journal of in Vitro Fertilization and Embryo Transfer, Vol. 6, No. 3, 1989
The Effect on Fertilization of Exposure of Mouse Oocytes to Dimethyl Sulfoxide: An Optimal Protocol MARTIN H. JOHNSON 1
Submitted: November 24, 1988 Accepted: January 23, 1989 (European Editorial Office)
during the freezing and thawing processes themselves or because of the adverse effects of temperature and/or cryoprotectant on oocytes in the prefreeze and postthaw periods. An improved understanding of the principles of freezing and thawing has enabled the development of successful protocols tailored to the size, membrane permeability, and internal composition of the oocyte (e.g., Refs. 6 and 11). As a result, cell death during the freezing process is being reduced (9). However, there is evidence that the conditions under which oocytes are prepared for, or recover from, the freezing process can lead to a number of changes that may affect adversely the cell's potential for normal development. Thus, transient cooling of the oocyte to 4~ in the absence of cryoprotectant reduces the fertilizability of the oocyte (2) and causes the disruption of its meiotic spindle, with attendant chromosomal dispersal and the potential for aneuploidy (3,12). Addition of the commonly used cryoprotectant dimethyl sulfoxide (DMSO) produces profound effects on the organization of the cytoskeleton and the metaphase plate, the nature, severity, and reversibility of which are temperature dependent (1,13). In this paper, a further adverse effect of DMSO is described, namely, a reduction in the sensitivity of the zona pellucida to digestion by chymotrypsin. This effect is associated with a reduced fertilization rate that reflects adverse effects of DMSO on both the zona and the cumulus mass. Conditions are defined under which these adverse effects may be minimized.
Mouse oocytes, with or without an intact cumulus mass, were exposed to various concentrations o f dimethyl sulfoxide (DMSO) at different temperatures for different periods o f time and using different protocols of DMSO addition and removal. The effect of these procedures on the chymotrypsin sensitivity o f the zona pellucida and the fertilizability o f the oocytes was then assessed. Some procedures were found to affect adversely both the zona pellucida and the cumulus mass, resulting in reductions in the fertilization rate. As a result o f both these and previously reported experiments (1-3), an optimal schedule is proposed for the handling o f mouse oocytes during cryopreservation, namely, to equilibrate cumulus-intact oocytes in 1.5 M DMSO precooled to 4~ prior to freezing, to remove DMSO at 4~ after thawing prior to restoring the oocytes to 37~ to loosen or remove the cumulus cells, and then to hold oocytes at 37~ for at least I hr to allow recovery o f the spindle prior to insemination. KEY WORDS: oocyte; zona pellucida; dimethyl sulfoxide; fertilization; cryopreservation; cumulus.
INTRODUCTION The successful long-term storage of gametes and the early preimplantation conceptus represents an important clinical advance in the treatment of infertility (4--6). However, this success is thus far limited to spermatozoa and early embryos. Unfertilized oocytes have proved in general much more difficult to cryopreserve in a demonstrably fertile state, some success having been achieved for the mouse (7,8) and, in only three fully reported cases, the human (9,10). Failure of oocyte cryopreservation could arise either because of irreversible damage
MATERIALS AND METHODS Oocyte Recovery and Handling Oocytes were recovered by superovulation of either MF 1 (laboratory Animal Centre) or (C57B16 z
1 Department of Anatomy, Downing Street, Cambridge CB2 3DY, UK. 0740-7769/89/06004)168506.00/09 1989PlenumPublishingCorporation
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169
OPTIMAL DMSO--g)OCYTE PROTOCOL
CBA) F~ mice (bred in the laboratory). Five to ten international units of pregnant mare serum (PMS) was followed 45-50 hr later by 5-10 IU of human chorionic gonadotropin (hCG; Intervet). Animals were killed three at a time 12.5 hr after the hCG injection, the oviducts transferred immediately to saline prewarmed to 37~ and the cumulus mass was then released immediately into M2 + bovine serum albumin (BSA) (14) at 37~ before the next group of animals was killed. When six to eight cumulus masses had been accumulated in one drop, they were, unless otherwise stated, exposed to 0.1 M hyaluronidase (Sigma) for 2-5 min. The oocytes were transferred to fresh M2 + BSA while they still had a residual corona of cumulus cells which dispersed in washing medium. Oocytes were pooled, counted, and distributed among control and experimental groups as indicated. All manipulative procedures were carried out on the heated stage of a Wild M5 dissecting microscope and embryo dishes were retained between manipulations at 37~ on warming blocks or in an incubator. These procedures were adopted to minimize temperature fluctuations and stress to the oocytes. For experimental treatments, oocytes were transferred at time zero to M2 + BSA in cavity blocks preequilibrated at the desired temperature and DMSO concentration according to schedules described under Results. Fertilization in Vitro Twelve hours after the administration of hCG to the oocyte donors, spermatozoa were recovered from CFLP males less than 6 months of age (Interfauna) that had last mated 48 hr previously. Fertilization in vitro was achieved by expelling spermatozoa from the vasa deferentia and cauda epididymides of a single male into 0.5 ml Whittingham's medium (15) containing 30 mg/ml BSA and incubat-
ing them at 37~ to capacitate until the cumulus-free oocytes were ready for insemination. The oocytes were then transferred to 1 ml of Whittingham's medium containing 30 mg/ml of BSA, and 50 to 75 ~l of spermatozoa were added to give a final concentration of motile spermatozoa of approximately 1-2 million/ml. Four hours after insemination, oocytes were rinsed free of spermatozoa, transferred to modified medium T6 + BSA (16), and scored 4-6 hr later for evidence of fertilization, as assessed by the presence of the second polar body and pronuclei. Cultures of fertilized eggs were then examined at intervals thereafter for evidence of cleavage to the two-cell stage and development to the blastocyst stage. Zona Resistance to Chymotrypsin Z o n a e were e x p o s e d to f r e s h l y p r e p a r e d ot-chymotrypsin (Sigma Type II, 0.001% in M2 + BSA) at 37~ Unless otherwise indicated, zonae expanded within 2 min of placing the oocytes in the enzyme, and within 5 min all oocytes could be sorted into two groups having either resistant (nonexpanded/nondissolved) or sensitive (expanded/ dissolved) zonae. These groups remained clearly distinguishable on further exposure to enzyme. Therefore, the proportion of oocytes with resistant zonae after 5 min of exposure to chymotrypsin is recorded under Results.
RESULTS Mouse oocytes were freed of their cumulus cells and placed for 30 min in one of a range of concentrations of DMSO preequilibrated at 37~ At the end of the incubation period, oocytes were rinsed for 30 min and then exposed to chymotrypsin. The
Table I. Effect on the Sensitivity of the Zona Pellucida to Chymotrypsin Digestion of Exposure of Cumulus-Free Mouse Oocytes to DMSO at 37~ for 30 min Percentage Concentration of DMSO (M)
No. of oocytes examined
Dead as result of procedure
Resistant to chymotrypsin
0 0.25 0.50 0.75 1.00 1.25 1.50
125 99 92 97 101 98 85
0 0 0 0 0 2 13
16 17 25 49 89 97 100
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proportion of oocytes with a zona pellucida resistant to chymotrypsin is indicated in Table I. It is clear that at concentrations of 0.5 M and above there is an increasing proportion of oocytes with resistant zonae. Exposure to concentrations of DMSO that are usually used for cryoprotectant function suppresssed zona removal almost entirely. Moreover, the same result was obtained when the washing period after removal from 1.5 M DMSO was increased to up to 2.5 hr with five changes of medium. Since it is not necessary for oocytes undergoing cryopreservation to be exposed to DMSO for prolonged periods prior to and following freezing, the effect of varying the duration of exposure to DMSO was investigated. The results in Table II show that even very short periods of exposure to DMSO at 37~ are sufficient to induce zona resistance to chymotrypsin. The protocols for addition and removal of DMSO to and from oocytes vary in different laboratories, some using stepwise rather than one-step addition and/or removal of DMSO. In Table III, the results indicate that the way in which the DMSO is removed does not appear to influence the induction of zona resistance to digestion, whereas a stepwise addition of DMSO did reduce, but did not eliminate, the effect. Stepwise addition and removal of the cryoprotectant are usually used to reduce the osmotic stress exerted on the oocytes. The effects of sucrose, a nonpenetrating osmotically active agent, on induction of zona resistance was therefore investigated. The results are shown in Table IV and show that a simple effect of osmotic stress on the oocytes is unlikely to explain the results obtained with DMSO. Effects of DMSO different from, or additional to, osmotic stress must be operating. Many protocols involve the addition and removal of DMSO not at 37~ but at room temperature. The consequences of varying the temperatures of addition and/or removal of DMSO were therefore exam-
ined, and the results are shown in Table V. The same induction of zona resistance is seen at room temperature as at 37~ However, when the temperature of both addition and removal was undertaken at 10 or 4~ no induction of zona resistance was observed. The addition of oocytes to DMSO at 4~ followed by their transfer to DMSO at 37~ prior to removal from DMSO did increase the proportion of zona resistant oocytes. Exposure to DMSO at 4~ for 120 min rather than 30 min induced a marginally higher level of zona resistance. During the exposure of oocytes to DMSO preequilibrated at different temperatures, different shrinkage patterns were observed. Thus, following the addition ofoocytes (N = 200) to DMSO at 37~ some appeared shrunken within 1 min, but by 5 min all had recovered their former volume. It took 7 min for complete recovery at room temperature (N = 200), 60 min for complete recovery at 10~ (N = 100), and over 120 min for recovery of 50 to 70% of oocytes at 4~ (N = 178). In the latter case, the recovery in many oocytes did not seem to be quite as complete as at higher temperatures. Cryopreservation usually involves oocytes with intact cumulus masses. A selected set of protocols was therefore applied to such oocytes, and the resuits are summarized in Table VI. The general pattern of results is similar for oocytes whether they are freed of their cumulus cells prior to or following DMSO exposure. However, it seems that the presence of the cumulus during exposure to DMSO does reduce slightly for each protocol studied the adverse effects of cryoprotectant. Resistance to chymotrypsin provides evidence of a change in the organization of the zona pellucida that can be indicative of a reduced fertilizability of the oocyte. In order to test directly whether such a reduction occurred, the fertilization and development rates of control oocytes were compared with those of oocytes that had been exposed to 1.5 M DMSO for 30 min at either 4 or 37~ and then re-
Table II. Effect of Duration of Exposure to 1.5 M DMSO at 37~ on Chymotrypsin Sensitivity of Zona Pellucida of Cumulus-Free Mouse Oocytes Percentage Duration of exposure (rain)
No. of oocytes
Dead as result of DMSO exposure
Resistant to chymotrypsin
0 5 15 30
115 76 80 105
0 3 5 13
16 100 99 100
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Table HI. Effect of Various Protocols for the Addition of Cumulus-Free Mouse Oocytes to, and their Subsequent Removal from, a 1.5 M DMSO Solution at 37~ on the Sensitivity of the Zona PeUucida to Chymotrypsin Percentage Protocol Addition
Removal
No. of oocytes
Dead as result of DMSO exposure
Resistant to chymotrypsin
1 step 1 step Stepwiseb Stepwiseb
1 step Stepwisea 1 step Stepwisea
208 278 240 248
19 9 3 2
99 98 57* 57*
a Removal of DMSO in 0.25 M steps of 5 min each commenced 5 min after the oocytes had been placed in 1.5 M DMSO. b Addition of DMSO in 0.25 M steps of 5 rain each; after 5 min in 1.5 M DMSO, removal procedure initiated. * Significantly different from one-step addition (P < 0.001; CHlZ).
moved from DMSO and washed extensively at either 4 or 37~ respectively, prior to insemination at 37~ Three experimental protocols were used. A protocol of exposure to DMSO, followed by washing and insemination of oocytes with an intact cumulus mass, resulted in reduced fertilization rates at both 4 and 37~ (Table VII, line 1). In contrast, if exposure of cumulus-intact oocytes to DMSO was followed by removal of the cumulus cells prior to insemination or if cumulus cells were removed prior to DMSO exposure, only those oocytes treated at 37~ showed reduced rates of fertilization (Table VII, lines 2 and 3). A protective effect of the cumulus cells on the 37~ DMSO-induced zona pellucida changes is again indicated from analysis of the resuits in Table VII, lines 2 and 3. The rate of development to blastocysts of those oocytes that did fertilize was not reduced significantly in any group or protocol studied. DISCUSSION The sensitivity of the mouse zona pellucida to digestion by chymotrypsin was described by Boldt
and Wolf (17) as providing an indication of an underlying change in its molecular organization that was also associated with a decreased incidence of sperm penetration. Typically, this kind of change is induced by fertilization (18) or parthenogenetic activation (19,20) of the oocyte, when it is known as "the zona reaction" and forms part of the block to polyspermy. However, increased resistance to both chymotrypsin and sperm penetration can also be seen in aged unfertilized oocytes ["zona hardening" (2,21-23)], after exposure of oocytes to phorbol esters (2,24,25), or after a period of 5 to 30 min at 4~ (2). The mechanism underlying the change in the zona pellucida in each of these cases is not established clearly, although it is assumed to represent a consequence of the release of the cortical granule contents, which then act enzymatically to modify and/or cross-link the zona glycoproteins (24-30). It is important to note that although activation, aging, phorbol esters, and cooling all lead to both chymotrypsin resistance and reduced fertilizability, it is not established formally that each of the two responses depends on a common underlying molecular mechanism. Indeed, it is clear that at
Table IV. Effect of Exposure to Sucrose (One-Step Addition and Removal at 37~ on Chymotrypsin Sensitivity of the Zona Pellucida of Cumulus-Free Mouse Oocytes Percentage Concentration of sucrose (M)
Duration of exposure (min)
No. of oocytes
Dead as result of sucrose exposure
Resistant to chymotrypsin
0 0.1 0.3 0.5 0.7 1.0 1.0
-30 30 30 30 30 5
50 100 50 50 60 50 70
0 0 0 0 0 100 0
22 25 22 26 13 -21
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Table V. Effect of Temperature on the Chymotrypsin Sensitivity of the Zona Pellucida of CumulusFree Mouse Oocytes Exposed to 1.5 M DMSO for 30 rain unless otherwise indicated (Compare with Values in Table III) Percentage Protocol Addition
Removal
1 step 20~ 1 step 20~ 1 step IO~ 1 step IO~ 1 step 4~ 1 step 4~ Stepwise 4~ 1 step 4~ c 1 step 4~ e
1 step 1 step 1 step 1 step 1 step 1 step 1 step 1 step 1 step
20~ a 20~ b
IO~ ~ lO~ b 4~ ~ 4~ b 4~ b 37~ d 4~ b
No. of oocytes
Dead as result of DMSO exposure
Resistant to chymotrypsin
80 70 153 160 245 683 305 291 78
0 0 1 4 7 11 14 7 20
95 91 21 25 13 11 17 31 25
a Hold at this temperature for 30 min, then take directly to chymotrypsin at 37~ b Hold at this temperature for 30 min, then take to 37~ for 30 min prior to exposure to chymotrypsin. r After 30 min at 4~ in DMSO, transfer to DMSO at 37~ for 30 min. d Hold at 37~ for 30 min before exposure to chymotrypsin. e Hold at 4~ for 120 min.
least some oocytes with zonae that are resistant to chymotrypsin can, nonetheless, be fertilized (2; this paper) and that induction of zona changes by different techniques does not result in identical functional and molecular modifications to zona proteins (25). The dissociation of chymotrypsin resistance from fertilizability could reflect either two independent responses to the same stimulus or a single response mechanism with different dose dependencies for the two bioassays used, chymotrypsin sensitivity being lost more easily than is sperm penetration. In this paper, the chymotrypsin sensitivity assay was used to investigate whether simple exposure of oocytes to DMSO had effects on the zona pellucida. Brief exposure to DMSO at concentrations required for effective cryoprotectant action was found to block zona lysis completely, but only if cumulusfree oocytes were added directly to DMSO at cryoprotectant levels at room temperature or
above. Retention of the cumulus and slower stepwise addition of the oocyte to the DMSO reduced (but did not elminate) the loss of chymotrypsin sensitivity. A complete loss of the effect on the zona was achieved by adding the oocytes to DMSO at 10 or 4~ but only if the DMSO was then removed at 4~ before restoration to 37~ Since we showed previously that transient exposure of oocytes to medium at 4~ (but not at 10~ lacking DMSO can itself result in a complete loss of chymotrypsin sensitivity (2), it is clear that exposure to precooled DMSO not only prevents the directly adverse effect seen at higher temperatures but also counteracts the adverse effect of exposure to 4~ alone. This result is somewhat analogous to the effects on the meiotic spindle of cooling, of DMSO, or of cooled DMSO (1,3). Thus, cooling led to the depolymerization of tubulin, causing the spindle to dismantle and the chromosomes to disperse, DMSO at room temper-
Table VI. Effect of Various Protocols of 1.5 M DMSO Exposure on the Chymotrypsin Sensitivity of the Zona Pellucida of Cumulus-Intact Mouse Oocytes Percentage Protocol Addition 1 step 37~ Stepwise 37~ 1 step 4~ 1 step 4~ b Stepwise 4~
Removal 1 step 1 step 1 step 1 step 1 step
37~ 37~ 4~ a 37~ c 4~
No. of oocytes
Dead as result of DMSO exposure
Resistant to chymotrypsin
374 174 424 165 204
3 2 0 3 1
84 34 8 24 12
" Hold at this temperature for 30 min, then take to 37~ for 30 min prior to exposure to chymotrypsin. b After 30 min at 4~ in DMSO, transfer to DMSO at 37~ for 30 min. c Hold at 37~ for 30 min before exposure to chymotrypsin.
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Table VII. Effect of Exposure to 1.5 M DMSO on Fertilization Rates of Mouse Oocytes Percentage fertilized (N) ProtocoV
Controls
After DMSO at 37~ b
After DMSO at 4~ b
1. Cumulus intact throughout
82 (129)
56** (191)
53** (220)
78 (142) 96 (97)
42* (294) 0.5* (193)
77 (319) 98 (103)
2. Strip cumulus after DMSO exposure but prior to insemination 3. Cumulus stripped at outset
a Each protocol was repeated twice and the results from each were combined. b Oocytes were placed directly into 1.5 MDMSO preequilibrated at 37 or 4~ After 30 min, oocytes were transferred directly into washing medium at 37 or 4~ After a further 30 min, oocytes were transferred to 37~ washing medium for 30 min prior to insemination. Control oocytes were taken through three 30-min incubations in washing medium at 37~ * Significantly different from control values (P ~< 0.01; X2). ** Significantly different from control values (P = 0.05; XZ).
ature or higher caused extensive polymerization of tubulin in the oocyte cytoplasm, leading secondarily to disorganization of the spindle and dispersion of the chromosomes, while the combination of DMSO and cooling led to stabilization of the spindle and its metaphase plate in a form more closely approximating a normal spindle than either condition alone. It is not clear from these studies why cooled DMSO is less deleterious for the zona pellucida than DMSO at room temperature or above. At lower temperatures, penetration of DMSO into the oocyte is slower than at 37~ or room temperature, as shown under Results by using the oocytes as "osmometers" and measuring the time they take to reexpand after initial exposure to DMSO. Oocytes at higher temperatures reexpanded rapidly, but those at 10~ reequilibrated fully after only 60 min, and even after 120 rain oocytes at 4~ had not recovered fully. While poor penetration of DMSO need not affect adversely its cryoprotectant action (6), it would have two consequences relevant to the results reported here. First, oocytes will be exposed to a more prolonged osmotic stress, which will lead to a more rapid and sustained loss of intracellular water (31). However, as the results of the experiments with sucrose show, simple osmotic stress does not result in a change of zona properties, indicating that alternative or additional factors must be involved. Second, at 4~ the interior of the cell will not be exposed to sudden high levels of DMSO; indeed the internal DMSO concentration achieved even after 120 min at 4~ is likely to be less than at 37~ That this lack of a sudden rise in intracellular
DMSO may be the more important reason for a reduced effect on the zona at low temperatures is indicated by the reduced zona effect observed (i) after the gradual addition of DMSO at 37~ (ii) after the addition of lower concentrations of DMSO at 37~ (iii) after holding oocytes at 4~ in DMSO for 120 min to allow gradual elevation of internal DMSO, and (iv) after retention of cumulus cells during exposure to DMSO at 37~ which might act to buffer the influx of the DMSO. It is not clear why or how a sudden influx of high levels of DMSO should elicit zona changes, but the stimulation of premature release of cortical granules would provide one explanation. There are indications that DMSO may affect the organization of the subcortical cytoskeletal mesh (13), perhaps allowing the cortical granules access to the oolemma, with the potential for fusion and the release and action of their contents. The effects of cooling alone to reduce zona sensitivity to chymotrypsin reported previously (2) were also not observed when DMSO was present. This result may be understood in terms of the more pronounced osmotic effects exerted by exposure to cooled DMSO. Thus, loss of intracellular water, which will reduce the contents of intracellular compartments, could balance any reduction in the capacity of those compartments as a result of the closer packing of membrane lipids at lower temperatures. Were such a rapid water loss not to occur, it is possible that compartment boundaries might shrink faster than their contents, so leading to leakiness. Leakage of cortical granule contents might affect the zona. The observations reported here could have im-
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portant practical consequence for approaches to cryopreservation of oocytes. Although the mouse oocyte may not be representative of all mammalian oocytes in its detailed cytoskeletal organization (32), there is no reason to believe that oocytes of other species, including man, may not show a similar response to DMSO. Indeed, schedules of exposure of human oocytes to DMSO that are found here to be nonoptimal for the mouse have been reported as resulting in comparable fertilization rates (33). We are currently examining this question directly on human oocytes. However, since exposure to DMSO at room temperature or above can reduce fertilization rates significantly, even after as little as 5 min, it may be prudent when attempting to cryopreserve oocytes to minimize this danger by precooling the DMSO. In the experiments reported here, the use of precooled DMSO removed entirely the effect on the zona, as assessed by both chymotrypsin resistance and fertilization rates. However, the fertilization studies did reveal a second potential problem with the use of DMSO as cryoprotectant, namely, an adverse effect on spermatozoal penetration through the cumulus mass. Thus, unless the cumulus was removed prior to insemination, even exposure to precooled DMSO was associated with reduced fertilization rates. In conclusion, our studies on both the cytoskeleton (1,3) and the fertilizability of oocytes lead us to the conclusion that the schedule with the least deleterious consequences for cryopreservation of mouse oocytes might be to equilibrate cumulusintact oocytes in precooled DMSO prior to freezing, to remove DMSO at 4~ after thawing prior to restoring the oocyte to 37~ to loosen or remove the cumulus cells, and then to hold oocytes at 37~ for at least 1 hr to allow recovery of the spindle prior to insemination. It is of interest to note that the most effective published protocol for cryopreserving mouse oocytes did in fact use the closest published protocol to that outlined above (7).
ACKNOWLEDGMENTS
I wish to thank Martin George, Brendan Doe, and Danny Hill for assistance and Maureen Wood, Brian Davis, Soo Pickering, and Peter Braude for valuable discussion of the manuscript drafts. This work was supported by a Medical Research Council program grant to MHJ and P. R. Braude.
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