Cryopreservation of unfertilized mouse oocytes: the effect of replacing sodium with choline in the freezing medium.
ABSTRACT Although embryo cryopreservation has become commonplace in many species, effective methods are not available for routine freezing of unfertilized eggs. Cryopreservation-induced damage may be caused by the high concentration of sodium ions in conventional freezing media. This study investigates the effect of a newly developed low-sodium choline-based medium (CJ2) on the ability of unfertilized, metaphase II mouse eggs to survive cryopreservation and develop to the blastocyst stage in vitro. Specifically, the effects of cooling to subzero temperatures, thawing rate, LN2 plunge temperature, and equilibration with a low-sodium medium prior to freezing are examined. In contrast to cooling to 23, 0, or -7.0 degreesC in a sodium-based freezing medium (ETFM), cooling in CJ2 had no significant negative effect on oocyte survival or development. Oocytes frozen in CJ2 survived plunging into LN2 from -10, -20, or -33 degreesC at significantly higher rates than oocytes frozen in ETFM. With the protocol used (1.5 M PrOH, 0.1 M sucrose, -0.3 C/min, plunging at -33 degreesC) rapid thawing by direct submersion in 30 degreesC water was more detrimental to oocyte survival than holding in air for 30 or 120 s prior to transfer to water. Equilibration of unfertilized oocytes with a low-sodium medium prior to cryopreservation in CJ2 significantly increased survival and blastocyst development. These results demonstrate that the high concentration of sodium in conventional freezing media is detrimental to oocyte cryopreservation and show that choline is a promising replacement. Reducing the sodium content of the freezing medium to a very low level or eliminating sodium altogether may allow oocytes and other cells to be frozen more effectively.
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ABSTRACT: A systematic approach was taken to assess and optimize a protocol for intracellular vitrification by introducing high concentrations of the cryoprotectant agent (CPA) ethylene glycol (EG) into unfertilized murine oocytes. The effects of EG on membrane integrity, microfilament organization, and developmental potential were evaluated. During exposure to 0.5-2 M EG, oocytes showed maximum shrinkage to 55.5% of the isotonic volume within the first minute and reexpanded to their initial volume within 15 min. Transferral of oocytes to higher concentrations of EG (4-8 M EG) for 1-5 min after 15 min of equilibration at 2 M EG was tolerated well. Microfilament organization appeared normal after this equilibration period. During prolonged exposure (> 5 min) to high concentrations of EG (> 4 M), membrane blebs were noticed on the surface of the cells, and microfilament distribution was disturbed. After treatment with 6 M EG and vitrification with 6 M EG + f2p40.5 M sucrose, there were no significant differences in development to the two-cell and blastocyst stages between CPA-treated, vitrified, and control oocytes. These results indicate that EG is an effective CPA for mouse oocyte vitrification protocols without any observed compromise in morphology and developmental functions.Biology of Reproduction 08/1996; 55(1):161-8. · 4.03 Impact Factor
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ABSTRACT: Rational design of a cryopreservation protocol was demonstrated by using theoretical models of the cryopreservation process to develop an optimal freezing protocol for mouse oocytes. A coupled mechanistic model of the processes of freeze-induced cell dehydration and intracellular ice formation was developed, and cryomicroscopical measurements of intracellular ice formation kinetics were used to determine biophysical parameters required by the model, and to test model predictions of the freezing behaviour of mouse oocytes. A simple phenomenological model for oocyte damage resulting from exposure to concentrated electrolyte and cryoprotectant solutions during cryopreservation was obtained by defining a cost function equal to the duration of the freezing protocol. A two-step freezing protocol was theoretically optimized by using a sequential simplex algorithm to minimize the cost function, subject to the constraint that the predicted probability of intracellular ice formation remain below 5%, yielding a putative optimum at the cooling rate B = 0.59 degrees C/min, and plunge temperature Tp = -67 degrees C. By systematically varying B and Tp about these values in experiments with mouse oocytes cryopreserved in 1.5 M dimethyl sulphoxide, the maximal recovery of intact oocytes with a normal morphology (82%) was obtained for B = 0.5 degrees C/min and Tp = -80 degrees C. Further evaluation of the fertilizability and developmental capacity of oocytes cryopreserved using the optimized protocol yielded cleavage to the 2-cell stage in 65% of oocytes inseminated, and blastocyst formation in 50% of these 2-cell embryos.Human Reproduction 07/1996; 11(6):1296-305. · 4.67 Impact Factor
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ABSTRACT: One-cell CF-1 x B6SJLF1/J embryos, which usually exhibit a 2-cell block to development in vitro, have been cultured to the blastocyst stage using CZB medium and a glucose washing procedure. CZB medium is a further modification of modified BMOC-2 containing an increased lactate/pyruvate ratio of 116, 1 mM-glutamine and 0.1 mM-EDTA but lacking glucose. Continuous culture of one-cell embryos in CZB medium allowed 83% of embryos to develop beyond the 2-cell stage of which 63% were morulae at 72 h of culture, but blastocysts did not develop. However, washing embryos into CZB medium containing glucose after 48 h of culture (3-4-cell stage) was sufficient to allow development to proceed, with 48% of embryos reaching the blastocyst stage by 96 h of culture. Exposure of embryos to glucose was only necessary from the 3-4-cell stage through the early morula stage since washing back into medium CZB without glucose at 72 h of culture still promoted the development of 50% of embryos to the blastocyst stage. The presence of glucose in this medium for the first 48 h of culture (1-cell to 4-cell stage) was detrimental to embryo development. Glutamine, however, exerted a beneficial effect on embryo development from the 1-cell to the 4-cell stage although its presence was not required for development to proceed during the final 48 h of culture. Blastocysts which developed under optimum conditions contained an average of 33.7 total cells. The in-vitro development of 1-cell embryos beyond the 2-cell stage in response to the removal of glucose and the addition of glutamine to the culture medium suggests that glucose may block some essential metabolic process, and that glutamine may be a preferred energy substrate during early development for these mouse embryos.J Reprod Fertil 08/1989; 86(2):679-88.