Abstract
Purpose
Our purpose was to assess the effect of cryopreservation on cytoskeleton of germinal vesicle (GV) mouse oocytes and determine whether irreversible spindle damage and related digyny associated with cryopreservation of metaphase II (MII) oocytes can be avoided.
Methods
The GV oocytes were cryopreserved using a slowcooling (0.5‡C/min) and slowthawing (8‡C/min) protocol in 1.5 M dimethylsulfoxide supplemented with 0.2 M sucrose and analyzed before and during fertilization by multiplelabel fluorescence and differential interference contrast microscopy techniques.
Results
When examined after in vitro maturation, the vast majority (>95%) of cryopreserved and control oocytes displayed normal microfilament and microtubule organization. With respect to barrelshaped spindle and normal chromosome alignment, no significant differences were observed between cryopreservation (78 and 86%, respectively) and control (85 and 95%, respectively) groups. In fertilization experiments, spindle rotation, formation of the second polar body, and pronuclear migration were displayed by similar percentages of cryopreserved (96, 94, and 37%, respectively) and control (98, 97, and 45%, respectively) oocytes, indicating normal functionality of the cytoskeleton during this period. However, pronuclear formation was significantly inhibited by cryopreservation (81%) compared with controls (100%). Regarding digyny and polyspermy, no significant increase was observed after cryopreservation (3 and 10%, respectively) compared with controls (3 and 6%, respectively).
Conclusions
Cryopreservation of mouse oocytes at the GV stage is particularly advantageous to circumvent the spindle damage and increased digyny noted after cryopreservation of MII oocytes.
Key words: cryopreservation, cytoskeleton, digyny, fertilization, oocyte, pronuclear formation
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References
- 1.Magistrini M, Szollosi D. Effects of cold and of isopropyl-N-phenylcarbamate on the second meiotic spindle of mouse oocytes. Eur J Cell Biol. 1980;22:699–707. [PubMed] [Google Scholar]
- 2.Aman RR, Parks JE. Effects of cooling and rewarming on the meiotic spindle and chromosomes of in vitro-matured bovine oocytes. Biol Reprod. 1994;50:103–110. doi: 10.1095/biolreprod50.1.103. [DOI] [PubMed] [Google Scholar]
- 3.Eroglu A, Toth TL, Toner M. Alterations of the cytoskeleton and polyploidy induced by cryopreservation of metaphase II mouse oocytes. Fertil Steril. 1998;69:944–957. doi: 10.1016/S0015-0282(98)00030-2. [DOI] [PubMed] [Google Scholar]
- 4.Vincent C, Garnier V, Heyman Y, Renard JP. Solvent effects on cytoskeletal organization and in-vivo survival after freezing of rabbit oocytes. J Reprod Fert. 1989;87:809–820. doi: 10.1530/jrf.0.0870809. [DOI] [PubMed] [Google Scholar]
- 5.Al-Hasani S, Diedrich K, Ven H, Reinecke A, Hartje M, Krebs D. Cryopreservation of human oocytes. Hum Reprod. 1987;2:695–700. doi: 10.1093/oxfordjournals.humrep.a136616. [DOI] [PubMed] [Google Scholar]
- 6.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. doi: 10.1002/mrd.1120160303. [DOI] [PubMed] [Google Scholar]
- 7.Carroll J, Warnes GM, Matthews CD. Increase in digyny explains polyploidy after in-vitro fertilization of frozen-thawed mouse oocytes. J Reprod Fert. 1989;85:489–494. doi: 10.1530/jrf.0.0850489. [DOI] [PubMed] [Google Scholar]
- 8.Maro B, Johnson MH, Webb M, Flach G. Mechanism of polar body formation in the mouse oocytes: an interaction between the chromosomes, the cytoskeleton and the plasma membrane. J Embryol Exp Morphol. 1986;92:11–32. [PubMed] [Google Scholar]
- 9.Schatten G, Simerly C, Schatten H. Microtubule configurations during fertilization, mitosis, and early development in the mouse and the requirement for egg microtubule-mediated motility during mammalian fertilization. Proc Natl Acad Sci USA. 1985;82:4152–4156. doi: 10.1073/pnas.82.12.4152. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Sathananthan AH, Trounson A, Freeman L, Brady T. The effects of cooling human oocytes. Hum Reprod. 1988;3:968–977. doi: 10.1093/oxfordjournals.humrep.a136827. [DOI] [PubMed] [Google Scholar]
- 11.Pickering SJ, Braude PR, Johnson MH, Cant A, Currie J. Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocytes. Fertil Steril. 1990;54:102–108. doi: 10.1016/s0015-0282(16)53644-9. [DOI] [PubMed] [Google Scholar]
- 12.Elst J, Abbeel E, Jacobs R, Wisse E, Steirteghem A. Effect of 1,2-propanediol and dimethylsulphoxide on the meiotic spindle of the mouse oocyte. Hum Reprod. 1988;3:960–967. doi: 10.1093/oxfordjournals.humrep.a136826. [DOI] [PubMed] [Google Scholar]
- 13.Mattson BA, Albertini DF. Oogenesis: Chromatin and microtubule dynamics during meiotic prophase. Mol Reprod Dev. 1990;25:374–383. doi: 10.1002/mrd.1080250411. [DOI] [PubMed] [Google Scholar]
- 14.Toth TL, Jones HW, Baka SG, Muasher S, Veeck LL, Lanzendorf S. Fertilization and in vitro development of cryopreserved human prophase I oocytes. Fertil Steril. 1995;61:891–894. doi: 10.1016/s0015-0282(16)56702-8. [DOI] [PubMed] [Google Scholar]
- 15.Elst JC, Nerinckx SS, Steirteghem AC. In vitro maturation of mouse germinal vesicle-stage oocytes following cooling, exposure to cryoprotectants and ultrarapid freezing: Limited effect on the morphology of the second meiotic spindle. Hum Reprod. 1992;7:1140–1446. doi: 10.1093/oxfordjournals.humrep.a137591. [DOI] [PubMed] [Google Scholar]
- 16.Baka SG, Toth TL, Veeck LL, Jones HW, Muasher SJ, Lanzendorf SE. Evaluation of the spindle apparatus of in vitro matured human oocytes following cryopreservation. Hum Reprod. 1995;7:1816–1820. doi: 10.1093/oxfordjournals.humrep.a136182. [DOI] [PubMed] [Google Scholar]
- 17.Park SE, Lee KA, Son WY, Ko JJ, Lee SH, Cha KY. Chromosome and spindle configurations of human oocytes matured in vitro after cryopreservation at the germinal vesicle stage. Fertil Steril. 1997;68:920–926. doi: 10.1016/S0015-0282(97)00365-8. [DOI] [PubMed] [Google Scholar]
- 18.Schroeder AC, Champlin AK, Mobraaten LE, Eppig JJ. Developmental capacity of mouse oocytes cryopreserved before and after maturation in vitro. J Reprod Fert. 1990;89:43–50. doi: 10.1530/jrf.0.0890043. [DOI] [PubMed] [Google Scholar]
- 19.Elst JC, Nerinckx SS, Steirteghem AC. 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. 1993;10:202–212. doi: 10.1007/BF01239222. [DOI] [PubMed] [Google Scholar]
- 20.Blerkom J, Davis PW. Cytogenetic, cellular, and developmental consequences of cryopreservation of immature and mature mouse and human oocytes. Microsc Res Tech. 1994;27:165–193. doi: 10.1002/jemt.1070270209. [DOI] [PubMed] [Google Scholar]
- 21.Ding J, Moor RM, Foxcroft GR. Effects of protein synthesis on maturation, sperm penetration, and pronuclear development in porcine oocytes. Mol Reprod Dev. 1992;33:59–66. doi: 10.1002/mrd.1080330109. [DOI] [PubMed] [Google Scholar]
- 22.Szollosi MS, Kubiak JZ, Debey P, Pennart H, Szollosi D, Maro B. Inhibition of protein kinases by 6-dimetylaminopurine accelerates the transition to interphase in activated mouse oocytes. J Cell Sci. 1993;104:861–872. doi: 10.1242/jcs.104.3.861. [DOI] [PubMed] [Google Scholar]
- 23.Schroeder AC, Eppig JJ. The developmental capacity of mouse oocytes that matured spontaneously in vitro is normal. Dev Biol. 1984;102:493–497. doi: 10.1016/0012-1606(84)90215-X. [DOI] [PubMed] [Google Scholar]
- 24.Schramm RD, Bavister BD. FSH-priming of rhesus monkeys enhances meiotic and developmental competence of oocytes in vitro. Biol Reprod. 1994;52:904–912. doi: 10.1095/biolreprod51.5.904. [DOI] [PubMed] [Google Scholar]