ISSN 2415-3060 (print), ISSN 2522-4972 (online)
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УЖМБС 2018, 3(3): 230–234
https://doi.org/10.26693/jmbs03.03.230
Biology

Ovarian Tissue Preservation after Various Cryopreservation Modes with the Help of DMSO

Trutaieva I. A., Kiroshka V. V., Gurina T. M., Bondarenko T. P., Filippov A. A.
Abstract

The purpose of the study was to investigate the follicular integrity and dynamics of thiobarbituric acid reactive substances (TBARS) accumulation in ovarian tissue fragments after cryopreservation depending on the final concentration of DMSO as well as the freezing mode. Material and methods. The research objects were the fragments of ovarian tissue. As cryoprotective agent (CPA) there was used dimethylsulfoxide (DMSO) in the concentrations of 1.5 M and 3,0 M in DMEM supplemented with 200 mM sucrose. The saturation was carried out step by step: 1.5 M (0 M → 1.5 M); 3.0 M in two stages (0 M → 1.5 M → 3.0 M). The tissue was incubated in CPA solution at 22°C for 30 min in each stage. Freezing was carried out in two modes. Mode 1: cooling at a rate of 1 deg / min to the start temperature of the initiation of crystal formation (IC), IC, further cooling at a rate of 1 deg / min to -40 °C and immersion into liquid nitrogen. Mode 2: cooling at a rate of 1 deg / min to -40 °C and subsequent immersion into liquid nitrogen. Samples were thawed in a water bath at 37 °C until the liquid phase appeared. The CPA was removed stepwise replacing the cryopreservation medium to DMEM solution containing mannitol. The number of normal and degenerative follicles was expressed as a percentage of the total number of follicles in the sample. The intensity of lipid peroxidation in the homogenate of cryopreserved tissue was assessed immediately after washing and after 3 and 24 hours of incubation in a DMEM medium in a CO2 incubator at 37 °C. Results and discussion. We noticed a decrease in the number of normal follicles by 20-30% at the stage of incubation after freezing under protection of 3.0 M DMSO (mode 2). This happened due to the presence of latent damage in the structure of ovarian tissue because of the toxic effect of this CPA. When 1.5 M DMSO was used, regardless of the freezing mode, such damage was absent, both after removal of the CPA and subsequent incubation of the tissue. It can be explained by uniform penetration of DMSO at 22°C throughout the tissue volume, which significantly reduces the effect of the mechanical factor of cell damage during crystallization. Conclusions. The obtained results indicate that the final concentration of DMSO determines the degree of damage to the ovarian tissue structure, depending on the freezing mode. It is necessary to select the cooling mode depending on the type and the final concentration of CPA while conducting cryopreservation of ovarian tissue.

Keywords: cryopreservation, ovarian tissue, DMSO, normal follicles, TBARS

Full text: PDF (Rus) 259K

References
  1. Amorim CA. Dolmans MM, David A, Jaeger J, Vanacker J, Camboni A, Donnez J, Van Langendonckt A. Vitrification and xenografting of human ovarian tissue. Fertil Steril. 2012; 98: 1291–8. https://www.ncbi.nlm.nih.gov/pubmed/22883570. https://doi.org/10.1016/j.fertnstert.2012.07.1109
  2. Demirci B, Lornage J, Salle B, Franck M, Frappart L, Guerin F. Follicular viability and morphology of sheep ovaries after exposure to cryoprotectant and cryopreservation with different freezing protocols. Fertil Steril. 2001; 75: 754–62. https://doi.org/10.1016/S0015-0282(00)01787-8
  3. Fabbri R, Pasquinelli G, Keane D, Magnani V, Paradisi R, Venturoli S. Optimization of protocols for human ovarian tissue cryopreservation with sucrose, 1,2-propanediol and human serum. Reproductive BioMedicine Online. 2010; 21: 819–28. https://doi.org/10.1016/j.rbmo.2010.07.008
  4. Gosden RG, Yin H, Bodine RJ, Morris GJ. Character, distribution and biological implications of ice crystallization in cryopreserved rabbit ovarian tissue revealed by cryo-scanning electron microscopy. Human Reproduction. 2010; 25 (2): 470–8. https://www.ncbi.nlm.nih.gov/pubmed/19933523. https://doi.org/10.1093/humrep/dep395
  5. Gurina TM, Vysekantsev IP, Babinets OM. Standartisation of cryopreservation process of SACCHAROMYCES BOULARDII yeasts for usage in collection and banks of industrial microorganism strains. Microbiologichny zhurnal. 2013; 75: 33–9. [Ukrainian]
  6. Kiroshka VV, Bozhkova YuO, Trutaieva IA, Gawas AA. Endokrynna funktsiia transplantativ ovarialnoi tkanyny v zalezhnosti vid intensyvnosti protsesiv perekysnoho okyslennia lipidiv pry ishemii. Fiziologichnyj Zhurnal. 2017; 63 (2): 46–55. [Ukrainian] https://doi.org/10.15407/fz63.02.046
  7. Kiroshka V, Trutaieva I, Bondarenko T. Efficiency of mannitol-supplemented medium during adding / removing ovarian tissue with penetrating cryoprotective agents. Cell & Tissue Banking. 2018 Mar;19 (1):123-32. https://www.ncbi.nlm.nih.gov/pubmed/28365880. https://doi.org/10.1007/s10561-017-9623-8
  8. Leibo SP. Cryopreservation of oocytes and embryos: optimization by theoretical versus empirical analysis. Theriogenology. 2008; 69: 37–47. https://www.ncbi.nlm.nih.gov/pubmed/18023472. https://doi.org/10.1016/j.theriogenology.2007.10.006
  9. Newton H, Fisher J, Arnold JRP, Pegg DEP, Faddy MJ, Gosden RG. Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation. Hum Reprod. 1998; 13: 376–80. https://www.ncbi.nlm.nih.gov/pubmed/9557842. https://doi.org/10.1093/humrep/13.2.376
  10. Rodrigues AP, Amorim CA, Costa SH, Matos MH, Santos RR, Lucci CM, Báo SN, Ohashi OM, Figueiredo JR. Cryopreservation of caprine ovarian tissue using dimethylsulphoxide and propanediol. Animal Reproduction Science. 2004; 84: 211–27. https://www.ncbi.nlm.nih.gov/pubmed/15302399. https://doi.org/10.1016/j.anireprosci.2003.12.003
  11. Shaw JM, Oranratnachai A, Trounson AO. Fundamental cryobiology of mammalian oocytes and ovarian tissue. Theriogenology. 2000; 53 (1): 59–72. https://www.ncbi.nlm.nih.gov/pubmed/10735062. https://doi.org/10.1016/S0093-691X(99)00240-X
  12. Uikli B. Jelektronnaja mikroskopija dlja nachinajushhih. M: Mir; 1975. 324 s. [Russian]
  13. Vladimirov YA, Archakov AI. Lipid peroxidation in biological membranes. Moscow: Nauka, 1972. 252 p. [Russian]
  14. Wang LH, Mullen SF, Li Y, Zhong JQ, Crister JK, Chen ZJ. Morphological and apoptotic comparison of primordial and primary follicles in cryopreserved human ovarian tissue. Reproduction in Domestic Animals. 2009; 44: 879–83. https://www.ncbi.nlm.nih.gov/pubmed/18992105. https://doi.org/10.1111/j.1439-0531.2008.01104.x
  15. Woods EJ, Benson JD, Agca Y, Critser JK. Fundamental cryobiology of reproductive cells and tissues. Cryobiology. 2004; 48: 146–56. https://www.ncbi.nlm.nih.gov/pubmed/15094091. https://doi.org/10.1016/j.cryobiol.2004.03.002