Purpose
The cryopreservation of mature metaphase II-stage mouse oocytes is associated with decreased fertilizability, spindle damage, and increased polyploidy. Therefore, we investigated the outcome of cryopreservation of immature germinal vesicle-stage mouse oocytes.
Methods
Oocytes were punctured from Graafian follicles in primed F1 hybrid mice and were then released into maturation medium containing the meiotic inhibitor dibutyryl cyclic AMP. Both slow and ultrarapid freezing protocols with dimethyl sulfoxide, 1,2-proponediol, or a mixture of both agents were tested. We recorded morphological survival rates, in vitro maturation rates, and two-cell and blastocyst formation rates. Each group of frozen oocytes was compared with both unfrozen germinal vesicle-stage oocytes and metaphase II-stage oocytes.
Results
An optimal cryosurvival rate of 78% was reached after ultrarapid freezing with 3 Mdimethyl sulfoxide followed by one-step dilution, but a decreased rate of twocell formation was observed. Freezing with a combination of dimethyl sulfoxide and 1,2-propanediol did not improve this fertilization-decreasing effect. Very low cryosurvival rates after freezing with 1,2-propanediol indicated its inappropriateness for ultrarapid freezing of immature oocytes. The rates of in vitro maturation were equivalent for frozen-thawed and freshly collected germinal vesicle-stage oocytes, independent of the freezing protocol used. We report, nevertheless, as a general characteristic for both slow and ultrarapid freezing of fully grown germinal vesicle-stage oocytes, that the in vitro development up to the blastocyst stage is inhibited despite full nuclear maturation.
Conclusion
We report that cryopreservation of immature germinal vesicle-stage oocytes is invariably associated with a low developmental capacity after fertilization. The rate of in vitro nuclear maturation did not equate with developmental competence. This in turn suggests the importance of cytoplasmic maturation for embryonic development.
Similar content being viewed by others
References
Whittingham DG: Fertilization in vitro and development to term of unfertilized mouse oocytes previously stored at − 196°C. J Reprod Fertil 1977;49:89–94
Glenister PH, Wood MJ, Kirby C, Whittingham DG: Incidence of chromosome anomalies in first-cleavage mouse embryos obtained from frozen-thawed oocytes fertilized in vitro. Gamete Res 1987;16:205–216
Johnson MH: The effect on fertilization of exposure of mouse oocytes to dimethylsulfoxide: An optimal protocol. J Vitro Fert Embryo Transfer 1989;6:168–175
Picketing SJ, Johnson MH: The influence of cooling on the organisation of the meiotic spindle of the mouse oocyte. Hum Reprod 1987;2:207–216
Johnson MH, Pickering: The effect of dimethylsulphoxide on the microtubular system of the mouse oocyte. Development 1987;100:313–324
Van der Elst J, Van den Abbeel E, Jacobs R, Wisse E, Van Steirteghem A: Effect of 1,2-propanediol and dimethylsulphoxide on the meiotic spindle of the mouse oocyte. Hum Reprod 1988;3:960–967
Pickering SJ, Cant A, Braude P, Currie J, Johnson MH: Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil Steril 1990;54:102–108
Al-Hasani S, Diedrich K, van der Ven H, Reinecke A, Hartje M, Krebs D: Cryopreservation of human oocytes. Hum Reprod 1987;2:695–700
Mandelbaum J, Junca AM, Plachot M, Alnot MO, Salat-Baroux J, Alvarez S, Tibi C, Cohen J, Debache C, Tesquier L: Cryopreservation of human embryos and oocytes. Hum Reprod 1988;3:117–119
Chen C: Pregnancy after human oocyte cryopreservation. Lancet 1986;1:884–886
Chen C: Oocyte freezing.In Clinical in Vitro Fertilisation, C Wood, A Trounson (eds). London, Springer-Verlag, 1989, pp 113–126
van Uem JF, Siebzehnrübl E, Schuh B, Koch R, Trotnow S, Lang N: Birth after cryopreservation of unfertilized oocytes. Lancet 1987;1:752–753
Schroeder AC, Eppig JJ: The developmental capacity of mouse oocytes that matured spontaneously in vitro is normal. Dev Biol 1984;102:493–497
Schroeder AC, Champlin AK, Mobraaten LE, Eppig JJ: Developmental capacity of mouse oocytes cryopreserved before and after maturation in vitro. J Reprod Fertil 1990;89:43–50
Trouson A, Peura A, Kirby C: Ultrarapid freezing: A new low-cost and effective method of cryopreservation. Fertil Steril 1987;48:843–850
Trouson A, Peura A, Freemann L, Kirby C: Ultrarapid freezing of early cleavage stage human embryos and eightcell mouse embroys. Fertil Steril 1988;49:822–826
Feichtinger W, Hochfellner Ch, Ferstl U: Clinical experience with ultrarapid freezing of embryos. Hum Reprod 1991;6:735–736
Gordts S, Roziers P, Campo R, Noto V: Survival and pregnancy outcome after ultrarapid freezing of human embryos. Fertil Steril 1990;53:469–472
Surrey ES, Quinn PJ: Successful ultrarapid freezing of unfertilized oocytes. J Vitro Fert and Embryo Transfer 1990;7:262–266
Hogan B, Costantini F, Lacy E: Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1986, pp 245–268
Wilson L, Quinn P: Development of mouse embryos cyropreserved by an ultra-rapid method of freezing. Hum Reprod 1989;4:86–90
Shaw JM, Kola I, MacFarlane DR, Trounson AO: An association between chromosomal abnormalities in rapidly frozen 2-cell mouse embryos and the ice-forming properties of the cryoprotective solution. J Reprod Fertil 1991;91:9–18
Shaw PW, Fuller BJ, Bernard A, Shaw RW: Vitrification of mouse oocytes: Improved rates of survival, fertilization, and development to blastocysts. Mol Reprod Dev 1991;29:373–378
Wood MJ, Carroll JC, Whittingham DG, Barros C, Candy C: Vitrification of mouse and hamster oocytes. Cryobiology 1991;28:515–516
Nakagata N: High survival rate of unfertilized mouse oocytes after vitrification. J Reprod Fertil 1989;87:479–483
Kono T, Kwon OY, Nakahara T: Development of vitrified mouse oocytes after in vitro fertilization. Cryobiology 1991;28:50–54
Van der Auwera I, Cornillie F, Ongkowidjojo R, Pijnenborg R, Koninckx PR: Cryopreservation of pronucleate mouse ova: Slow versus ultrarapid freezing. Hum Reprod 1990;5:619–621
Todorow SJ, Siebzehnrübl ER, Koch R, Wildt L, Lang N: Comparative results on survival of human and animal eggs using different cryoprotectants and freeze-thawing regimens. I. Mouse and hamster. Hum Reprod 1989;4:805–811
Todorow SJ, Siebzehnrübl ER, Spitzer M, Koch R, Wildt L, Lang N: Comparative results on survival of human and animal eggs using different cryoprotectants and freeze-thawing regimens. II. Human. Hum Reprod 1989;4:812–816
Downs SM, Schroeder AC, Eppig JJ: Developmental capacity of mouse oocytes following maintenance of meiotic arrest in vitro. Gamete Res 1986;15:305–316
Wassarman PM, Fujiwara K: Immunofluorescent antitubulin staining of spindles during meiotic maturation of mouse oocytesin vitro. J Cell Sci 1978;29:171–188
Albertini DF: Novel morphological approaches for the study of oocyte maturation. Biol Reprod 1984;30:13–28
Rime H, Jessus C, Ozon R: Distribution of microtubules during the first meiotic cell division in the mouse oocyte: Effect of Taxol. Gamete Res 1987;17:1–13
Alexandre A, Mulnard J: Time-lapse cinematography study of the germinal vesicle behaviour in mouse primary oocytes treated with activators of protein kinases A and C. Gamete Res 1988;21:359–365
Van Blerkom J: Maturation at high frequency of germinal vesicle-stage mouse oocytes after cryopreservation: Alterations in cytoplasmic, nuclear, nucleolar and chromosomal structure and organisation associated with vitrification. Hum Reprod 1989;4:883–898
Stricker SA, Schatten G: The cytoskeleton and nuclear disassembly during germinal vesicle breakdown in starfish oocytes. Dev Growth Differ 1991;33:163–171
Schroeder AC, Downs SM, Eppig JJ: Factors affecting the developmental capacity of mouse oocytes undergoing maturation in vitro. Ann NY Acad Sci 1988;541:197–204
Shaw JM, Trounson AO: Parthenogenetic activation of unfertilized mouse oocytes by exposure to 1,2-propanediol is influenced by temperature, oocyte age and cumulus removal. Gamete Res 1989;24:269–279
Van der Elst J, Van den Abbeel E, Nerinckx S, Van Steirteghem AC: Parthenogenetic activation pattern and microtubular organization of the mouse oocyte after exposure to 1,2-propanediol. Cryobiology 1992;29:549–562
Carroll J, Whittingham DG, Wood MJ, Telfer E, Gosden RG: Extra-ovarian production of mature viable mouse oocytes from frozen primary follicles. J Reprod Fertil 1990;90:321–327
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Van Der Elst, J.C., Nerinckx, S.S. & Van Steirteghem, A.C. Slow and ultrarapid freezing of fully grown germinal vesicle-stage mouse oocytes: Optimization of survival rate outweighed by defective blastocyst formation. J Assist Reprod Genet 10, 202–212 (1993). https://doi.org/10.1007/BF01239222
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01239222