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Factors affecting low temperature survival of mammalian oocytes
THERIOGENOLOGY
FACTORS
AFFECTING LOW TEMPERATURE OF MAMMALIAN OOCYTES
SURVIVAL
John E. Parks and Nancy A. Ruffing Department of Animal Science Cornell University Ithaca, New York 14853 ABSTRACT The ability to cryopreserve mammalian oocytes effectively would greatly increase their availability for a broad range of reproductive technologies. Oocytes have been frozen using both equilibrium and non-equilibrium approaches originally developed for mammalian cleavage-stage embryos, but rates of fertilization and development are typically much lower than with unfrozen oocytes. Production of live young from frozen oocytes of domestic animals has not been reported. Sensitivity of cytoskeletal elements, the meiotic spindle and other components of the cortical ooplasm to chilling and cryoprotective agents may contribute to the limited success in oocyte cryopreservation. Oocytes are also subject to physical events during freezing which influence cell survival. Estimates of biophysical parameters which influence the osmotic and cryobiological responses of oocytes are becoming available and may be useful for developing freezing protocols. KEYWORDS:
oocyte,
cryopreservation,
mammals
Introduction The availability of viable, developmentally competent oocytes has been critical to recent progress in the development of in vitro fertilization (IVF), embryo culture and related reproductive technologies in mammals. However, the relatively short fertile life of mammalian oocytes is a limiting factor in the implementation of many in vitro methodologies. This limitation could be overcome largely through cryopreservation, as practiced currently with mammalian embryos from many species (1). Oocyte cryopreservation
Acknowledgements Original studies referred to in this manuscript were supported by U.S.D.A. grant 8937240-4773. The authors wish to thank Drs. P. L. Steponkus and R. E. Pitt for assistance with these studies.
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would permit women anticipating loss of gonadal function through surgery or other therapies to maintain their reproductive potential and circumvent the many ethical and legal questions surrounding Female germ lines of laboratory and human embryo cryostorage. domestic species could be preserved indefinitely or banked for use in research and commercial applications ranging from providing hamster ova for sperm penetration assays to research and development related to cloning and genetic engineering. The germ including genetically engineered plasm of valuable females, individuals and endangered species, could be salvaged after loss of normal fertility or even death. However, it is generally recognized that the developmental competence of cryopreserved mammalian oocytes is extremely limited (reviewed in l-3). Cryopreserved oocytes from several species have been fertilized in vitro (see 2), but live births have been reported only for the mouse, rabbit and human (4-10). The purpose of this paper is to examine special features of mammalian oocytes which may contribute to their refractoriness to cryopreservation, the procedural steps where damage may occur and recent efforts to develop effective cryopreservation protocols. Special Features
Of Mammalian
Oocytes
Besides being extraordinarily large cells, oocytes are characterized by several other unique features as they develop from the germinal vesicle stage to metaphase II. Key among these relevant to cryopreservation are structural and molecular features of the cytoskeleton, meiotic spindle, oolemma and zona pellucida, and the distribution of cortical granules (CGs) in the ooplasm. Arrangements of actin-containing microfilaments are found in the perinuclear and cortical ooplasm which are involved in organellar distribution during oocyte maturation, surface deformations associated with polar body extrusion, motility of microvilli and sperm engulfment (11,12). Oocytes arrested at metaphase II contain a peripheral, anastral spindle apparatus, with microtubules extending from each pole to the kinetochores of bivalent chromosomes. Chromosomes at the metaphase plate and pericentriolar material associated with each pole act as microtubule organizing centers for tubulin polymerization during spindle formation (11,12). During metaphase II, Golgi-derived CGs are positioned immediately subjacent to the oolemma where they are
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triggered to undergo exocytosis at fertilization. Release of CG contents consequently alters the zona pellucida (cortical granule reaction), creating a block to polyspermy (12). The oolemma of the mammalian oocyte is covered with numerous microvilli containing an actin filament core. Very little is known about the biochemical composition of the bilayer of this membrane; however, one might speculate that the oolemma is easily destabilized, as it is poised for fusion with the fertilizing sperm and CGs. Effects
Of Cryoprotective
Agents
On Mammalian
Oocytes
Cryoprotective agents (CPAs) are needed for freezing tissues and cells; but at the molar concentrations required, CPAs may also cause cell damage due to osmotic effects or chemical toxicity. Several studies have demonstrated CPA effects on cytoskeletal elements of mammalian oocytes. Brief exposure of mouse oocytes to 1.5 M dimethylsulfoxide (DMSO) promoted tubulin polymerization, causing abnormal aster formation at the spindle poles and in the cytoplasm (13). Asters remained after prolonged exposure, the spindle was reduced and chromosomes became dispersed. Spindles reassembled after DMSO removal. Propylene glycol (PG) has been associated with parthenogenetic activation of mouse oocytes (14). Exposure of rabbit oocytes to 1.5 M PG but not DMSO reversibly depolymerized the cortical band of filamentous actin in mature oocytes while both CPAs had effects on tubulin-containing structures similar to those observed with mouse oocytes (9). Complete disappearance of abnormal asters after CPA removal required 3 hr at 370C and even then, spindles were not always restored. It was suggested that PG depolymerization of actin may actually provide some advantage during cryopreservation by increasing tolerance to osmotic shrinkage. Disruption of cortical microfilaments in mouse oocytes caused by DMSO has been reported recently (15). Addition of 1.5 M DMSO to mouse oocytes at 370C also resulted in zona hardening as assessed by resistance to chymotrypsin (16), but stepwise addition at 4oC minimized this effect. Changes in the zonae of mouse and human oocytes by CPAs have been correlated with CG release and reduced fertilization rates (17-19). Provided procedures are used to minimize osmotic stress, the integrity of the oolemma is not generally lost when conventional cryopreservation protocols requiring l-2 M CPAs are used;
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however, detrimental effects on maturation and fertilization following CPA treatment are sometimes observed. Immature bovine oocytes appear to be more sensitive to CPAs than in vitro matured Exposure to 10% (v/v) DMSO, ethylene glycol (IVM) oocytes. (EG), glycerol (GLY) or PG for 5 min reduced the ability of immature oocytes to progress to metaphase II by 43-74% (20). However, IVF and cleavage rates of >70% were obtained with immature oocytes exposed to 1.5 M DMSO/O.S M sucrose and IVM oocytes exposed to either 2.0 M DMSO or PG (21). In our experience, IVM bovine oocytes, held in 1.5 M PG at 350C for 10 min to reach osmohave IVF rates ranging from 75-781 .of those of tic equilibrium, control oocytes whether the PG is removed stepwise in 1 .O and 0.5 M PG solutions or in a single step using 0.75 M sucrose. High IVF rates (93% of control values) were also obtained with IVM oocytes equilibrated with 1.5 M GLY followed by one-step removal in sucrose. t Effects
Of Chilling
And Freezing
Mammalian
Oocytes
Specific detrimental effects caused by cooling oocytes below body temperature have been reported for many species. Most striking are effects on the second meiotic spindle where microtubules are disrupted or disassembled, apparently due to tubulin depolymerization (22-26). Most mouse oocytes exposed to 250C had abnormal or missing spindles after only 10 min, with 8% having dispersed chromosomes (22). Oocytes similarly exposed to 4oC had no normal spindles after 10 min, the majority of spindles were completely disassembled within 20 min and 14% of oocytes had dispersed chromosomes. Spindle appearance returned to normal after rewarming to 370C for 60 min. However, spindles in less than half of human oocytes exposed to room temperature for 10 min returned to a normal appearance after 4 hr at 370C (23). The inability of human oocytes to reassemble the spindle apparatus was attributed to a lack of pericentriolar material required for nucleating polymerization. We have recently demonstrated that the meiotic spindle of IVM bovine oocytes is sensitive to chilling, with spindles reduced or absent in >50% of oocytes exposed to 250C for 1 min and absent in all oocytes held at 4oC for >20 min (25). Experiments to evaluate spindle reassembly by bovine oocytes upon rewarming are in progress. 1 Ruffhg and Parks, in preparation.
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Cooling also alters the zona pellucida (27,28) resulting in decreased sensitivity to chymotrypsin and reduced fertilization rates in the mouse. This effect appears to be indirect, caused by CG exocytosis leading to a premature cortical granule reaction. Chilling alone does not appear to compromise seriously the integrity of the oolemma in most species, yet subtle changes in membrane structure and function may occur during cooling (29,30) due to thermotropic phase transitions of polar lipids and consequent lateral phase separation of membrane components. Altered distribution of bilayer lipids and proteins may result in membrane destabilization upon rewarming (3 1). The overall effect of chilling oocytes is equivocal for the mouse, as the reduced fertilization rate and development to the blastocyst stage attributed to the premature cortical granule reaction at 4oC (27) has not been observed by others at 0-8oC (5,32-35). Sensitivity of domestic animal oocytes to chilling appears to be more significant. Immature porcine oocytes do not survive cooling (30) and ovine oocytes cooled to 20-300C during IVM and fertilized in vivo exhibit a 4-fold or greater reduction in their ability to develop to the blastocyst stage (36). We have observed a 13% reduction in IVF rate of IVM bovine oocytes held at OoC for 2 min and a 28% reduction after 30 min.1 The physical events which occur during freezing and the theoretical basis for how these events influence cell survival are firmly established (37,38). During conventional or equilibrium cryopreservation, extracellular ice formed spontaneously or by seeding creates a gradient in the chemical potential for water, favoring exosmosis from the unfrozen, intracellular compartment (39,40). Cooling rates too rapid to permit osmotic equilibrium result in intracellular ice formation (IIF). The extent of cell injury due to IIF varies depending upon the method of cryopreservation, but with conventional protocols, IIF is associated with damage to the plasma membrane and is generally presumed to be lethal to the cell. Very slow cooling rates are also detrimental as they result in prolonged exposure to elevated solute concentrations. In addition, freezing and thawing tend to exacerbate the effects of chilling and CPAs on cytoskeletal elements, the meiotic spindle, CGs and the zona pellucida (32,41-43).
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Progress
In Oocyte Cryopreservation
Even though servation procedures,
oocytes are especially sensitive to cryopreprogress has been made in oocyte cryopreser-
vation primarily by adapting protocols used successfully with preimplantation mammalian embryos. Early reports have been reviewed recently (l-3). More recent efforts are summarized in Table 1 and include both equilibrium and non-equilibrium approaches (39,40). Variations in experimental detail are numerous, but a survey of results provides several overall impressions. First, the preferred TABLE
1. Recent
publications
on cryopreservation
of mammalian
oocytes.
Methoda
Reference
mature
slow rapid ultrarapid vitrification
5,35,41,44,45 32,35,45-47 42,48,49 6,33,50-53
immature
slow ultrarapid vitrification
54-56 57 58
Rat
immature
rapid
59
Hamster
mature
slow rapid ultrarapid vitrification
45,60 61 62 52
Rabbit
mature
slow rapid
63 9,10,64
Human
mature
slow ultrarapid
44,63,65,66 65
cow
mature immature
rapid rapid
67 68
Sheep
immature
slow
69
Pig
immature
rapid vitrification
30 70
SDecies Mouse
aAccording
64
to (71).
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CPAs for freezing oocytes are DMSO and PG for conventional protocols and ultrarapid freezing while vitrification solutions are generally modifications of those of Rall and Fahy (72). Oocytes maintain their morphological integrity under a variety of freeze/thaw regimens and encouraging fertilization and cleavage rates have been reported. However, when results from the literature are evaluated on the basis of viable offspring/oocyte frozen, it is clear that only a small percentage of frozen oocytes can be fertilized and develop to term in most species regardless of the developmental stage or method used. Better results are possible with mouse oocytes, for which success rates of 15-30% viable offspring/oocyte frozen have been obtained with both conventional protocols and vitrification. Novel approaches to oocyte cryopreservation, such as freezing primary follicles (54,55) or use of anti-freeze proteins (70), may offer some advantages with further development of procedures. Finally, very little progress in freezing domestic animal oocytes has been reported (30,67-70). Fundamental
Cryobiology
Of Mammalian
Oocytes
To date, attempts to develop cryopreservation procedures for mammalian oocytes have been based primarily on empirical approaches. However, information on biophysical parameters of oocytes required to predict optimal freezing protocols has begun to accumulate. These parameters include plasma membrane permeability to water (hydraulic conductivity or Lp) and CPAs, the temperature-dependence of these permeabilities and the probability of IIF and mean IIF temperature as a function of cooling rate and the suspending medium. In 1963, Mazur (37) demonstrated how these parameters could be used to model thermodynamically a cell’s volumetric response to freezing. Mazur’s model has since been refined so that underlying assumptions more accurately reflect actual rather than ideal conditions (34,73). In addition, the ability of predictive models to simulate a cell’s response to specific cryopreservation conditions has been confirmed using data collected on cell types ranging from plant protoplasts to mammalian embryos (38,74-76). The large size and sphericity of mammalian oocytes makes them amenable to osmometric and cryobiologic studies. The volumetric response to anisotonic conditions has been studied in oocytes from including the mouse (77), hamster (78) and cow several species,
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(79,80). The plot of equilibrium oocyte volume vs. l/osmolality of the suspending medium is linear for each species examined, indicating that oocytes behave as ideal osmometers. Extrapolating this relationship to infinite osmolalities gives an estimate of the fractional volume of the oocyte that is occupied by non-osmotically active solids (FVb). Estimates of FVb for mammalian oocytes range from 0.17 to 0.32 (77-80). Knowing the value of FVb, one can estimate the oocyte’s L, using cell volume measurements collected as a function of elapsed time during osmotic equilibration. Mean L, values between 0.36 and 0.54 pl(min*atm) have been reported for mouse oocytes (77,81) and immature bovine oocytes (79). Higher mean LP values ranging from 0.65 to 0.84 p/(min*atm) have been measured in hamster (78), human (81) and IVM bovine oocytes (80). Most investigators have limited their study to Lp at room temperature (20-230C); however, Lp is temperature-dependent and a quantitative understanding of this dependence below OoC is needed to improve the accuracy of the predictions of thermodynamic models (73,82). Although activation energy (Ea) estimates made above OoC for mouse (77) and bovine oocytesl’ may be used to provide crude estimates of subzero L, values, direct measurements of Lp at subzero temperatures are only available for mouse oocytes (0.04 p/(min*atm); 82). Quantitative information on CPA permeability of oocytes is limited. Based on visual estimates of the time required to re-expand to their original isotonic volume, we have concluded that IVM bovine oocytes are more permeable to DMSO, EG and PG (5-10 min to equilibrate) than GLY (30 min). 1 Similar results with EG and GLY were reported with bovine embryos (83), but according to Leibo (84), DMSO penetrates bovine blastocysts more slowly than GLY. More striking developmental effects on CPA permeability have been quantified for mouse oocytes (85). Within 3 h after fertilization, GLY permeability increased more than 3-fold from 7.3 to 24.7 x 10_5p/(min*atm). Studies of oocyte cryobiology have been completed for several species. The mean IIF temperature of oocytes in salt solutions has been reported consistently as -12 to -130C with the probability of IIF increasing dramatically from 0 to 100% over a narrow range of cooling rates (hamster, 3 to 8oC/min; bovine, 1 to 16oC/min; mouse, 1 to SoC/min) (34,78,79,86,87). The probability of IIF in
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mouse oocytes has also been studied under isothermal conditions the percentages of (34). After 200 set at -3.8, -6.4 or -7.7oC, oocytes with IIF were 0, 50 and 60%, respectively. In the presence of various CPAs, the mean IIF temperature becomes significantly lower and highly dependent upon the cooling rate (78,79,87,88). Currently, estimates of the most critical biophysical parameters are available only for the mouse. Information on human and domestic animal oocytes necessary for the prediction of improved cryopreservation protocols is still lacking. Certain parameters with similar values for species examined to date, such as mean IIF temperature, may require less rigorous study in all species. However, accurate estimates of Lp, Es and other parameters will be essential for developing models to predict optimal cryopreservation procedures. CONCLUSIONS Mammalian oocytes are characterized by several unique features which increase their susceptibility to damage during cryopreservation procedures. Disassembly of the meiotic spindle due to chilling, CPAs or freezing is of concern because of the potential for causing chromosomal abnormalities such as aneuploidy and polyploidy (41,89). Depolymerization of actin-containing microfilaments by CPAs and freezing may perturb cytoskeletal arrangements critical to the progression of meiosis and normal fertilization. Release of CGs during cooling, CPA addition or freezing appear to cause a premature cortical granule reaction leading to either a block to fertilization or an impaired block to polyspermy. The potential for plasma membrane damage and loss of cell viability during freezing per se is well established. As a single cell, the oocyte is likely to be more vulnerable to any environmental challenge than the multi-cellular preimplantation embryo, and so each procedural step during cryopreservation probably contributes, separately or cumulatively, to the low developmental potential of frozen oocytes. Improvements in cryopreservation procedures may require special steps to minimize the forms of injury, caused by chilling sensitivity and CPA toxicity, which are only indirectly related to freezing. Additional fundamental studies to clarify mechanisms of cryoinjury and establish optimal freezing protocols are needed to improve upon purely empirical approaches to oocyte cryopreservation.
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63. Siebzehnrubl, E.R., Todorow, S., van Uem, J., Koch, R., Wildt, L. and Lang, N. Cryopreservation of human and rabbit oocytes and one-cell embryos: a comparison of DMSO and propanediol. Hum. Reprod. q:312317 (1989). 64. Heyman, Y., Campion, E., Chesne, P. and Renard, J. Cryopreservation of recipient oocytes for nuclear transfer in the rabbit species. Cryobiology u: in press (1991). H. and Krebs, D. Cryopre65. Al-Hasani, S., Diedrich, K., VanDerVen, servation of human and rabbit oocytes. Cryobiology a:584-585 (1988). 66. Hunter, J., Bernard, A., Fuller, B. and Shaw R. Investigation of glycerol and DMSO as cryoprotectants for unfertilized murine and human oocytes: fertilization and development following fertilization. Cryobiology 28: in press (1991). 67. Lim, J.M., Fukui, Y. and Ono, H. The post-thaw developmental capacity of frozen bovine oocytes following in vitro maturation and fertilization. Theriogenology s:1225-1235 (1991). 68. Schellander, K., Brackett, B.G., Fuhrer, F. and Schleger, W. In vitro fertilization of frozen thawed cattle oocytes. 11th Int. Cong. Anim. Reprod. A.I. (Dublin) a:349 (1988). 69. Sulieman, D., Jamal-Aldein, N. and Ridha-Albarzanchi, M. The effect of thawing rate on in-vitro maturation of frozen-thawed ovine oocytes. Hum. Reprod. q(Supp1.): 118 (1990). 70. Rubinsky, B., Arav, A. and Devries, A.L. using directional cooling and antifreeze u:93-106 (1991). 71. Pomp, D. and Critser, J. Cryopreservation Atlas of Science, pp. 40-46 (1988).
Cryopreservation glycoproteins. of mammalian
of oocytes Cryo-Letters
embryos.
72. Rall, W. and Fahy, G. Ice-free cryopreservation of mouse -1960C by vitrification. Nature 3_!_3:573-575 (1985).
h
IS1
embryos
at
73. Pitt, R.E., Myers, S.P., Lin, T.T. and Steponkus, P.L. Subfreezing volumetric behavior and stochastic modeling of intracellular ice formation in Drosophila melanogaster embryos. Cryobiology 2:72-86 (1991). 74. Mazur, P., Rall, W.F. and Leibo, likelihood of intracellular freezing 213 (1984). 75. Mazur,
72
P. and Schneider,
U. Osmotic
S.P. Kinetics of water loss and the in mouse ova. Cell Biophys. 6:197responses
of preimplantation
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THERIOGENOLOGY
and bovine embryos 8:259-285 (1986).
and their
cryobiological
implications.
Cell Biophys.
76. Pitt, R.E. and Steponkus, P.L. Quantitative analysis of the probability of intracellular ice formation during freezing of isolated protoplasts. Cryobiology x:44-63 (1989). 77. Leibo, S.P. Water permeability and its activation energy of fertilized unfertilized mouse ova. J. Membrane Biol. u:179-188 (1980). 78. Shabana, M. and McGrath, J.J. Cryomicroscope dynamic modeling of the freezing of unfertilized x:338-354 (1988).
and
investigation and thermohamster ova. Cryobiology
79. Myers, S.P., Lin, T., Pitt, R.E. and Steponkus, P.L. Cryobehavior immature bovine oocytes. Cryo-Letters 8:260-275 (1987). 80. Ruffing, N.A., Parks, J.E., Pitt, R.E. and Steponkus, P.L. developmental stage on the osmotic properties of bovine Cryobiology u:675 (1990).
of
Effect of oocytes.
81. Bernard, A., McGrath, J.J., Fuller, B.J., Imoedemhe, D. and Shaw, R.W. Osmotic response of oocytes using a microscope diffusion chamber: A preliminary study comparing murine and human ova. Cryobiology 25:495501 (1988). 82. Toner, M., Cravalho, E.G. and Armant, D.R. estimated transmembrane potential during freezing Membrane Biol. m:261-272 (1990). 83. Szell, A., Shelton, and cattle embryos.
Water transport and of mouse oocytes. J.
J.N. and Szell, K. Osmotic characteristics Cryobiology =:297-301 (1989).
84. Leibo, S.P. A one-step in situ dilution method embryos. Cryo-Letters &:387-400 (1983). 85. Jackowski, S.C., Leibo, fertilized and unfertilized
of sheep
for frozen-thawed
bovine
S.P. and Mazur, P. Glycerol permeabilities of mouse ova. J. Exp. Zool. m:329-341 (1980).
86. Chandrasekaran, M., Pitt, R.E., Parks, J.E. and Steponkus, Stochastic behavior of intracellular ice formation in isolated protoplasts and mature bovine oocytes. Cryobiology =:676 (1990).
P.L. rye
87. Ruffing, N.A. and Parks, J.E. Cryobehavior of mature and fertilized bovine oocytes. McGrath, J.J. and Cravalho, E.G. Microscopic observation of intracellular ice formation in unfertilized mouse ova as a function of cooling rate. Cryobiology u:257-271 (1978). observation 88. Leibo, S.P., McGrath, J.J. and Cravalho, E.G. Microscopic of intracellular ice formation in unfertilized mouse ova as a function of cooling rate. Crybiology 15:257-271 (1978). 89. Kola, I., Kirby, C., Shaw, J., Davey, A. and Trounson, A. Vitrification of mouse oocytes results in aneuploid zygotes and malformed fetuses. Teratology B:467-474 (1988).
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FACTORS
AFFECTING LOW TEMPERATURE OF MAMMALIAN OOCYTES
SURVIVAL
John E. Parks and Nancy A. Ruffing Department of Animal Science Cornell University Ithaca, New York 14853 ABSTRACT The ability to cryopreserve mammalian oocytes effectively would greatly increase their availability for a broad range of reproductive technologies. Oocytes have been frozen using both equilibrium and non-equilibrium approaches originally developed for mammalian cleavage-stage embryos, but rates of fertilization and development are typically much lower than with unfrozen oocytes. Production of live young from frozen oocytes of domestic animals has not been reported. Sensitivity of cytoskeletal elements, the meiotic spindle and other components of the cortical ooplasm to chilling and cryoprotective agents may contribute to the limited success in oocyte cryopreservation. Oocytes are also subject to physical events during freezing which influence cell survival. Estimates of biophysical parameters which influence the osmotic and cryobiological responses of oocytes are becoming available and may be useful for developing freezing protocols. KEYWORDS:
oocyte,
cryopreservation,
mammals
Introduction The availability of viable, developmentally competent oocytes has been critical to recent progress in the development of in vitro fertilization (IVF), embryo culture and related reproductive technologies in mammals. However, the relatively short fertile life of mammalian oocytes is a limiting factor in the implementation of many in vitro methodologies. This limitation could be overcome largely through cryopreservation, as practiced currently with mammalian embryos from many species (1). Oocyte cryopreservation
Acknowledgements Original studies referred to in this manuscript were supported by U.S.D.A. grant 8937240-4773. The authors wish to thank Drs. P. L. Steponkus and R. E. Pitt for assistance with these studies.
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would permit women anticipating loss of gonadal function through surgery or other therapies to maintain their reproductive potential and circumvent the many ethical and legal questions surrounding Female germ lines of laboratory and human embryo cryostorage. domestic species could be preserved indefinitely or banked for use in research and commercial applications ranging from providing hamster ova for sperm penetration assays to research and development related to cloning and genetic engineering. The germ including genetically engineered plasm of valuable females, individuals and endangered species, could be salvaged after loss of normal fertility or even death. However, it is generally recognized that the developmental competence of cryopreserved mammalian oocytes is extremely limited (reviewed in l-3). Cryopreserved oocytes from several species have been fertilized in vitro (see 2), but live births have been reported only for the mouse, rabbit and human (4-10). The purpose of this paper is to examine special features of mammalian oocytes which may contribute to their refractoriness to cryopreservation, the procedural steps where damage may occur and recent efforts to develop effective cryopreservation protocols. Special Features
Of Mammalian
Oocytes
Besides being extraordinarily large cells, oocytes are characterized by several other unique features as they develop from the germinal vesicle stage to metaphase II. Key among these relevant to cryopreservation are structural and molecular features of the cytoskeleton, meiotic spindle, oolemma and zona pellucida, and the distribution of cortical granules (CGs) in the ooplasm. Arrangements of actin-containing microfilaments are found in the perinuclear and cortical ooplasm which are involved in organellar distribution during oocyte maturation, surface deformations associated with polar body extrusion, motility of microvilli and sperm engulfment (11,12). Oocytes arrested at metaphase II contain a peripheral, anastral spindle apparatus, with microtubules extending from each pole to the kinetochores of bivalent chromosomes. Chromosomes at the metaphase plate and pericentriolar material associated with each pole act as microtubule organizing centers for tubulin polymerization during spindle formation (11,12). During metaphase II, Golgi-derived CGs are positioned immediately subjacent to the oolemma where they are
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triggered to undergo exocytosis at fertilization. Release of CG contents consequently alters the zona pellucida (cortical granule reaction), creating a block to polyspermy (12). The oolemma of the mammalian oocyte is covered with numerous microvilli containing an actin filament core. Very little is known about the biochemical composition of the bilayer of this membrane; however, one might speculate that the oolemma is easily destabilized, as it is poised for fusion with the fertilizing sperm and CGs. Effects
Of Cryoprotective
Agents
On Mammalian
Oocytes
Cryoprotective agents (CPAs) are needed for freezing tissues and cells; but at the molar concentrations required, CPAs may also cause cell damage due to osmotic effects or chemical toxicity. Several studies have demonstrated CPA effects on cytoskeletal elements of mammalian oocytes. Brief exposure of mouse oocytes to 1.5 M dimethylsulfoxide (DMSO) promoted tubulin polymerization, causing abnormal aster formation at the spindle poles and in the cytoplasm (13). Asters remained after prolonged exposure, the spindle was reduced and chromosomes became dispersed. Spindles reassembled after DMSO removal. Propylene glycol (PG) has been associated with parthenogenetic activation of mouse oocytes (14). Exposure of rabbit oocytes to 1.5 M PG but not DMSO reversibly depolymerized the cortical band of filamentous actin in mature oocytes while both CPAs had effects on tubulin-containing structures similar to those observed with mouse oocytes (9). Complete disappearance of abnormal asters after CPA removal required 3 hr at 370C and even then, spindles were not always restored. It was suggested that PG depolymerization of actin may actually provide some advantage during cryopreservation by increasing tolerance to osmotic shrinkage. Disruption of cortical microfilaments in mouse oocytes caused by DMSO has been reported recently (15). Addition of 1.5 M DMSO to mouse oocytes at 370C also resulted in zona hardening as assessed by resistance to chymotrypsin (16), but stepwise addition at 4oC minimized this effect. Changes in the zonae of mouse and human oocytes by CPAs have been correlated with CG release and reduced fertilization rates (17-19). Provided procedures are used to minimize osmotic stress, the integrity of the oolemma is not generally lost when conventional cryopreservation protocols requiring l-2 M CPAs are used;
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however, detrimental effects on maturation and fertilization following CPA treatment are sometimes observed. Immature bovine oocytes appear to be more sensitive to CPAs than in vitro matured Exposure to 10% (v/v) DMSO, ethylene glycol (IVM) oocytes. (EG), glycerol (GLY) or PG for 5 min reduced the ability of immature oocytes to progress to metaphase II by 43-74% (20). However, IVF and cleavage rates of >70% were obtained with immature oocytes exposed to 1.5 M DMSO/O.S M sucrose and IVM oocytes exposed to either 2.0 M DMSO or PG (21). In our experience, IVM bovine oocytes, held in 1.5 M PG at 350C for 10 min to reach osmohave IVF rates ranging from 75-781 .of those of tic equilibrium, control oocytes whether the PG is removed stepwise in 1 .O and 0.5 M PG solutions or in a single step using 0.75 M sucrose. High IVF rates (93% of control values) were also obtained with IVM oocytes equilibrated with 1.5 M GLY followed by one-step removal in sucrose. t Effects
Of Chilling
And Freezing
Mammalian
Oocytes
Specific detrimental effects caused by cooling oocytes below body temperature have been reported for many species. Most striking are effects on the second meiotic spindle where microtubules are disrupted or disassembled, apparently due to tubulin depolymerization (22-26). Most mouse oocytes exposed to 250C had abnormal or missing spindles after only 10 min, with 8% having dispersed chromosomes (22). Oocytes similarly exposed to 4oC had no normal spindles after 10 min, the majority of spindles were completely disassembled within 20 min and 14% of oocytes had dispersed chromosomes. Spindle appearance returned to normal after rewarming to 370C for 60 min. However, spindles in less than half of human oocytes exposed to room temperature for 10 min returned to a normal appearance after 4 hr at 370C (23). The inability of human oocytes to reassemble the spindle apparatus was attributed to a lack of pericentriolar material required for nucleating polymerization. We have recently demonstrated that the meiotic spindle of IVM bovine oocytes is sensitive to chilling, with spindles reduced or absent in >50% of oocytes exposed to 250C for 1 min and absent in all oocytes held at 4oC for >20 min (25). Experiments to evaluate spindle reassembly by bovine oocytes upon rewarming are in progress. 1 Ruffhg and Parks, in preparation.
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Cooling also alters the zona pellucida (27,28) resulting in decreased sensitivity to chymotrypsin and reduced fertilization rates in the mouse. This effect appears to be indirect, caused by CG exocytosis leading to a premature cortical granule reaction. Chilling alone does not appear to compromise seriously the integrity of the oolemma in most species, yet subtle changes in membrane structure and function may occur during cooling (29,30) due to thermotropic phase transitions of polar lipids and consequent lateral phase separation of membrane components. Altered distribution of bilayer lipids and proteins may result in membrane destabilization upon rewarming (3 1). The overall effect of chilling oocytes is equivocal for the mouse, as the reduced fertilization rate and development to the blastocyst stage attributed to the premature cortical granule reaction at 4oC (27) has not been observed by others at 0-8oC (5,32-35). Sensitivity of domestic animal oocytes to chilling appears to be more significant. Immature porcine oocytes do not survive cooling (30) and ovine oocytes cooled to 20-300C during IVM and fertilized in vivo exhibit a 4-fold or greater reduction in their ability to develop to the blastocyst stage (36). We have observed a 13% reduction in IVF rate of IVM bovine oocytes held at OoC for 2 min and a 28% reduction after 30 min.1 The physical events which occur during freezing and the theoretical basis for how these events influence cell survival are firmly established (37,38). During conventional or equilibrium cryopreservation, extracellular ice formed spontaneously or by seeding creates a gradient in the chemical potential for water, favoring exosmosis from the unfrozen, intracellular compartment (39,40). Cooling rates too rapid to permit osmotic equilibrium result in intracellular ice formation (IIF). The extent of cell injury due to IIF varies depending upon the method of cryopreservation, but with conventional protocols, IIF is associated with damage to the plasma membrane and is generally presumed to be lethal to the cell. Very slow cooling rates are also detrimental as they result in prolonged exposure to elevated solute concentrations. In addition, freezing and thawing tend to exacerbate the effects of chilling and CPAs on cytoskeletal elements, the meiotic spindle, CGs and the zona pellucida (32,41-43).
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Progress
In Oocyte Cryopreservation
Even though servation procedures,
oocytes are especially sensitive to cryopreprogress has been made in oocyte cryopreser-
vation primarily by adapting protocols used successfully with preimplantation mammalian embryos. Early reports have been reviewed recently (l-3). More recent efforts are summarized in Table 1 and include both equilibrium and non-equilibrium approaches (39,40). Variations in experimental detail are numerous, but a survey of results provides several overall impressions. First, the preferred TABLE
1. Recent
publications
on cryopreservation
of mammalian
oocytes.
Methoda
Reference
mature
slow rapid ultrarapid vitrification
5,35,41,44,45 32,35,45-47 42,48,49 6,33,50-53
immature
slow ultrarapid vitrification
54-56 57 58
Rat
immature
rapid
59
Hamster
mature
slow rapid ultrarapid vitrification
45,60 61 62 52
Rabbit
mature
slow rapid
63 9,10,64
Human
mature
slow ultrarapid
44,63,65,66 65
cow
mature immature
rapid rapid
67 68
Sheep
immature
slow
69
Pig
immature
rapid vitrification
30 70
SDecies Mouse
aAccording
64
to (71).
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CPAs for freezing oocytes are DMSO and PG for conventional protocols and ultrarapid freezing while vitrification solutions are generally modifications of those of Rall and Fahy (72). Oocytes maintain their morphological integrity under a variety of freeze/thaw regimens and encouraging fertilization and cleavage rates have been reported. However, when results from the literature are evaluated on the basis of viable offspring/oocyte frozen, it is clear that only a small percentage of frozen oocytes can be fertilized and develop to term in most species regardless of the developmental stage or method used. Better results are possible with mouse oocytes, for which success rates of 15-30% viable offspring/oocyte frozen have been obtained with both conventional protocols and vitrification. Novel approaches to oocyte cryopreservation, such as freezing primary follicles (54,55) or use of anti-freeze proteins (70), may offer some advantages with further development of procedures. Finally, very little progress in freezing domestic animal oocytes has been reported (30,67-70). Fundamental
Cryobiology
Of Mammalian
Oocytes
To date, attempts to develop cryopreservation procedures for mammalian oocytes have been based primarily on empirical approaches. However, information on biophysical parameters of oocytes required to predict optimal freezing protocols has begun to accumulate. These parameters include plasma membrane permeability to water (hydraulic conductivity or Lp) and CPAs, the temperature-dependence of these permeabilities and the probability of IIF and mean IIF temperature as a function of cooling rate and the suspending medium. In 1963, Mazur (37) demonstrated how these parameters could be used to model thermodynamically a cell’s volumetric response to freezing. Mazur’s model has since been refined so that underlying assumptions more accurately reflect actual rather than ideal conditions (34,73). In addition, the ability of predictive models to simulate a cell’s response to specific cryopreservation conditions has been confirmed using data collected on cell types ranging from plant protoplasts to mammalian embryos (38,74-76). The large size and sphericity of mammalian oocytes makes them amenable to osmometric and cryobiologic studies. The volumetric response to anisotonic conditions has been studied in oocytes from including the mouse (77), hamster (78) and cow several species,
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(79,80). The plot of equilibrium oocyte volume vs. l/osmolality of the suspending medium is linear for each species examined, indicating that oocytes behave as ideal osmometers. Extrapolating this relationship to infinite osmolalities gives an estimate of the fractional volume of the oocyte that is occupied by non-osmotically active solids (FVb). Estimates of FVb for mammalian oocytes range from 0.17 to 0.32 (77-80). Knowing the value of FVb, one can estimate the oocyte’s L, using cell volume measurements collected as a function of elapsed time during osmotic equilibration. Mean L, values between 0.36 and 0.54 pl(min*atm) have been reported for mouse oocytes (77,81) and immature bovine oocytes (79). Higher mean LP values ranging from 0.65 to 0.84 p/(min*atm) have been measured in hamster (78), human (81) and IVM bovine oocytes (80). Most investigators have limited their study to Lp at room temperature (20-230C); however, Lp is temperature-dependent and a quantitative understanding of this dependence below OoC is needed to improve the accuracy of the predictions of thermodynamic models (73,82). Although activation energy (Ea) estimates made above OoC for mouse (77) and bovine oocytesl’ may be used to provide crude estimates of subzero L, values, direct measurements of Lp at subzero temperatures are only available for mouse oocytes (0.04 p/(min*atm); 82). Quantitative information on CPA permeability of oocytes is limited. Based on visual estimates of the time required to re-expand to their original isotonic volume, we have concluded that IVM bovine oocytes are more permeable to DMSO, EG and PG (5-10 min to equilibrate) than GLY (30 min). 1 Similar results with EG and GLY were reported with bovine embryos (83), but according to Leibo (84), DMSO penetrates bovine blastocysts more slowly than GLY. More striking developmental effects on CPA permeability have been quantified for mouse oocytes (85). Within 3 h after fertilization, GLY permeability increased more than 3-fold from 7.3 to 24.7 x 10_5p/(min*atm). Studies of oocyte cryobiology have been completed for several species. The mean IIF temperature of oocytes in salt solutions has been reported consistently as -12 to -130C with the probability of IIF increasing dramatically from 0 to 100% over a narrow range of cooling rates (hamster, 3 to 8oC/min; bovine, 1 to 16oC/min; mouse, 1 to SoC/min) (34,78,79,86,87). The probability of IIF in
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mouse oocytes has also been studied under isothermal conditions the percentages of (34). After 200 set at -3.8, -6.4 or -7.7oC, oocytes with IIF were 0, 50 and 60%, respectively. In the presence of various CPAs, the mean IIF temperature becomes significantly lower and highly dependent upon the cooling rate (78,79,87,88). Currently, estimates of the most critical biophysical parameters are available only for the mouse. Information on human and domestic animal oocytes necessary for the prediction of improved cryopreservation protocols is still lacking. Certain parameters with similar values for species examined to date, such as mean IIF temperature, may require less rigorous study in all species. However, accurate estimates of Lp, Es and other parameters will be essential for developing models to predict optimal cryopreservation procedures. CONCLUSIONS Mammalian oocytes are characterized by several unique features which increase their susceptibility to damage during cryopreservation procedures. Disassembly of the meiotic spindle due to chilling, CPAs or freezing is of concern because of the potential for causing chromosomal abnormalities such as aneuploidy and polyploidy (41,89). Depolymerization of actin-containing microfilaments by CPAs and freezing may perturb cytoskeletal arrangements critical to the progression of meiosis and normal fertilization. Release of CGs during cooling, CPA addition or freezing appear to cause a premature cortical granule reaction leading to either a block to fertilization or an impaired block to polyspermy. The potential for plasma membrane damage and loss of cell viability during freezing per se is well established. As a single cell, the oocyte is likely to be more vulnerable to any environmental challenge than the multi-cellular preimplantation embryo, and so each procedural step during cryopreservation probably contributes, separately or cumulatively, to the low developmental potential of frozen oocytes. Improvements in cryopreservation procedures may require special steps to minimize the forms of injury, caused by chilling sensitivity and CPA toxicity, which are only indirectly related to freezing. Additional fundamental studies to clarify mechanisms of cryoinjury and establish optimal freezing protocols are needed to improve upon purely empirical approaches to oocyte cryopreservation.
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