The Cytotoxic Effect of Cryoprotective Agents on in vitro Fertilization Rates of Mammalian Oocytes

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Melanie McDowell
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1 Cean A. et al./scientific Papers: Animal Science and Biotechnologies, 2013, 46 (2) The Cytotoxic Effect of Cryoprotective Agents on in vitro Fertilization Rates of Mammalian Oocytes Ada Cean 1,2,*, Ivan Alexandra 3, Ilie Daniela 2, Nicolae Păcală 1 1 Banat s University of Agricultural Sciences and Veterinary Medicine Timişoara, Calea Aradului, No. 119, , Romania 2 Research and Development Station for Bovine - Arad, , Bodrogului 32, Arad, Romania 3 University of Medicine and Pharmacy "Victor Babes" Timisoara, , Eftimie Murgu 2, Romania Abstract Oocyte cryopreservation has become an integral part of Assisted Reproductive Technologies (ART). The aim of our study was to evaluate the cytotoxic effect of penetrating and non-penetrating cryoprotector agents used in oocyte cryopreservation media. We exposed mature and immature mice oocytes to different concentrations of cryoprotectors (penetrating and non-penetrating). As penetrating cryoprotectors we used glycerol, dimethyl sulfoxide (DMSO) and 1,2 propandiol, in concentrations of 1.5 moles L -1, 3 moles L -1, 5 moles L -1, 7 moles L -1. As non-penetrating cryoprotectors we used sucrose, trehalose and Ficoll 70 in concentration of 10%, 15% and 20%. The oocytes were exposed to cryoprotective solutions for 3, 5 and 10 minutes after which were subjected to in vitro fertilization procedure. Our result showed that 15% non-penetrating cryoprotectors and 3 moles L -1 penetrating cryoprotector has no effect on in vitro fertilization rates of oocytes, when the exposure time did not exceed 5 minutes. Ficoll 70 cannot be used in controlled freezing of the oocytes since the exposure of oocytes more than 5 minutes is impairing the fertilization process. At 1.5 moles L -1 penetrating cryoprotector the in vitro fertilization rates were not affected, regardless of the exposure time meaning that glycerol, DMSO and 1,2 propandiol can be used for controlled freezing of oocytes. Keywords: cryopreservation, cryoprotectors, cytotoxicity, oocyte, in vitro fertilization 1. Introduction Oocyte cryopreservation is much difficult, than embryo, sperm or cells, mainly because of the fact that the oocytes are one of the biggest cells in the organism and in freezing size is a disadvantage in respect to survival after thawing [1]. Although, there were significant improvements made there is the necesity of establishing a freezing protocol expecialy designed for oocytes [2]. Reducing of the toxicity of the cryoprotecors and the evaluation of the effect of the cryoprotectors on to the physiology of the cells is also an insufficient researched problem in oocytes cryobiology [3]. * Corresponding author: Cean A., telea_ada@animalsci-tm.ro The aim of our study was to evaluate the cytotoxic effect of penetrating and non-penetrating cryoprotector agents used in oocyte cryopreservation media. 2. Materials and methods All chemicals were purchased from Sigma, unless other producer is specified. Biologic material: as biological material we used mouse females, age 2 months, and mouse males with proved fertility. The animals were maintained in 12 hours light and 12 hours dark period. Oocyte recovery: For mature oocyte recovery the females were stimulated using 5 IU ecg (Folligon, Intervet) si 5 UI hcg (Chorulon, Intervet), hormone administration was performed at 48h interval, the last injection was performed 98

Immature oocytes were recovered directly form ovaries by follicle dissection. For maturation oocytes were cultured 17h in MEM-α (Invitrogen) supplemented with 5% FBS.

2 with 13 hours before recovery. The recovery of the oocytes was performed as described by A. Naghy (2003). For immature oocyte the females were stimulated with 5IU ecg, the hormone administration was performed with 48h before recovery. Immature oocytes were recovered directly form ovaries by follicle dissection. For maturation oocytes were cultured 17h in MEM-α (Invitrogen) supplemented with 5% FBS. The oocytes were recovered in HTF (Human Tubal Fluid) with 25mM HEPES. In vitro fertilization: Sperm was recovered from epididymis tail, and capacitated by incubation at 37 C, in HTF for 1 h. For in vitro fertilization a 1-2.5x 10 6 ml -1 concentration of sperm was used. After 4-6 h of coculture with sperm, the oocytes were washed and placed in KSOM-AA for overnight culture (37 C, 5%CO2, humidified atmosphere). The fertilisation rate was assessed the next morning. Evaluation of cytotoxic effect of the cryoprotectors: the oocytes were exposed, before in vitro fertilization, at different concentrations of penetrating (glycerol, DMSO, 1,2 propandiol) and unprenetrating (sucrose, trehalose and Ficoll 70), the concentration and exposure time are presented in table 1: Table 1. Cryoprotectors concentration and exposure time Exposure to cryoprotectors for 3, 5 and 10 min. Unpenetrating Concentration (%) Penetrating Concentration (mol. L -1 ) Sucrose Glycerol Trehalose DMSO Ficoll ,2 Propandiol Results and discussion Results after exposing the mature oocytes to unpenetrating cryoprotectors Figure. 1. In vitro fecundation rates after mature oocytes exposure to sucrose Figure.3. In vitro fecundation rates after mature oocytes exposure to Ficoll Except thehalose, 5 min. exposure (fig. 2), none of the oocytes were fecundated when exposed to 20% unpenetrating cryoprotector were tested (fig. Figure.2. In vitro fecundation rates after mature oocytes exposure to trehalose 1, 2, 3). Ficoll seems to be the most toxic since at 10 minutes exposure no fecundation was obtained. For sucrose, when exposure time was 10 min. the fecundation decreased significantly 24% at 10% concentration and 22.67% at 20% concentration. Trehalose seems to be the leas toxic for mature oocytes, at 10% and 15% concentration the was no decrees in fecundation rate, regardless of the exposure time, and even at 20% concentration in media we obtained fecundation, when exposure was short (3 min.) (fig. 3). 99

Results after exposing the immature oocytes to unpenetrating cryoprotectors Figure.4.

In vitro fertilization rates after immature oocytes exposure to trehalose Figure 6.

of 20% no maturation was obtained (fig. 4, 5, and 6). For sucrose the maturation rate was 75% and 70.97% when 10% and 15 % sucrose was used, for 3 minutes.

When exposure time increased at 5 minutes a slow decrease of the maturation and fecundation rate was obtained, with no statistical difference (p>0.05) (fig.

by in vitro condition since at 15% sucrose the fertilization rate was 42.11%. When trehalose was used the results were similar with sucrose.

4% when 10% Ficoll was used, and 31.04% when 15% Ficoll was used.

3 Results after exposing the immature oocytes to unpenetrating cryoprotectors Figure.4. In vitro fertilization rates after immature oocytes exposure to sucrose Figure.5. In vitro fertilization rates after immature oocytes exposure to trehalose Figure 6. In vitro fertilization rates after immature oocytes exposure to Ficoll When immature oocytes were exposed to unpenetrating cryoprotector at a concentration of 20% no maturation was obtained (fig. 4, 5, and 6). For sucrose the maturation rate was 75% and 70.97% when 10% and 15 % sucrose was used, for 3 minutes. The fecundation rate was 47.62% and 50% respectively. When exposure time increased at 5 minutes a slow decrease of the maturation and fecundation rate was obtained, with no statistical difference (p>0.05) (fig.4). A significant decrease was notice in fecundation rates of in vitro matured oocytes exposed for 10 minutes to 10 % sucrose but it most likely is caused by in vitro condition since at 15% sucrose the fertilization rate was 42.11%. When trehalose was used the results were similar with sucrose. Ficoll exposure seems to be the most cytotoxic for immature oocytes since at 10 minutes exposure the maturation rated decreased significantly at 34.4% when 10% Ficoll was used, and 31.04% when 15% Ficoll was used. In respect to the fertilization rates there were no fertilized oocytes when the exposure time was 10 minutes at 10% and 15 % Ficoll. The cryoprotector concentration and the duration of time spend by the embryo, and oocytes into the vitrification solution is negatively correlated with embryo survival rate [5] A Ficoll is used in later years in vitrification of embryos, in order to reduce the concentrations of penetrating cryoprotectants. The cryopreservation studies using Ficoll as unpenetrating cryoprotector in embryo freezing shows that Ficoll has a low toxicity for embryos [6]. 100

Results after exposing the mature oocytes to penetrating cryoprotectors Figure 7.

In vitro fecundation rates after mature oocytes exposure to 1,2 propandiol Figure. 8.

unpenetrating cryoprotectors the best results were obtained with glycerol (fig. 7).

significantly regardless of the exposure time (fig. 7, 8 and 9).

when exposure was 3 minutes. Interestingly was that 1.

4 Results after exposing the mature oocytes to penetrating cryoprotectors Figure 7. In vitro fecundation rates after mature oocytes exposure to glycerol Figure 9. In vitro fecundation rates after mature oocytes exposure to 1,2 propandiol Figure. 8. In vitro fecundation rates after mature oocytes exposure to DMSO From the unpenetrating cryoprotectors the best results were obtained with glycerol (fig. 7). From 5 moles L -1 concentration the fertilization rates started to decrease significantly regardless of the exposure time (fig. 7, 8 and 9). At 7 moles L -1 cryoprotector, except glycerol, there were no oocytes fertilized even when exposure was 3 minutes. Interestingly was that 1.5 moles L -1 and 3 M concentrations seem to have no effect on fertilization rates of the mature mouse oocytes. Results after exposing the immature oocytes to penetrating cryoprotectors Figure10. In vitro fertilization rates after immature oocytes exposure to glycerol Figure 11. In vitro fertilization rates after immature oocytes exposure to DMSO 101

Figure 12. In vitro fertilization rates after imature oocytes exposure to 1,2 propandiol Immature oocytes seem to be more sensitive to exposure at penetrating cryoprotector.

Exposure of the immature oocytes led to a decrease in maturation rate and fertilization (18.75%) starting at 1.5 moles L -1, when exposure was 10 min. (fig. 10).

5 Figure 12. In vitro fertilization rates after imature oocytes exposure to 1,2 propandiol Immature oocytes seem to be more sensitive to exposure at penetrating cryoprotector. At 7 moles L -1 penetrating cryoprotector no maturation was achieved, regardless of the exposure time (fig. 10, 11, and 12). Exposure of the immature oocytes led to a decrease in maturation rate and fertilization (18.75%) starting at 1.5 moles L -1, when exposure was 10 min. (fig. 10). Although at 7 moles L -1 there were no fertilized oocytes for DMSO, it seems to be less toxic for immature oocytes when compared with glycerol since maturation rates after exposure are satisfactory after exposure to concentrations of 1.5 and 3 mol. L -1, at 5 moles L - 1, no fertilization was achieved after 3 minutes of exposure. Studies performed by Huang et. al. (2006), showed that the oocytes exposed to DMSO 1M, have no decreases in fertility rates. When the concentration of DMSO is higher, 2M the fecundity rates decrease, but the studies of Huang et. al. (2006), showed that DMSO has a stabilization role for the microtubules in oocytes cytoplasm [7]. Interestingly is the effect of 1.2 popandiol, which showed no toxic effect at concentrations from 1.5 to 5 moles L -1, regardless of the exposure time. The toxic effect of the cryoprotectors, on the viability of the cells is stated in specialty literature but the mechanism thru which it accts is not fully known. Huang et. al. (2006) has performed studies that are suggesting that the toxicity of the cryoprotectors is due to specific and nonspecific effects like dehydration and chemical cytotoxicity [7]. Mouse oocytes exposed for 20 minutes at a 1 mole L -1 sucrose solution have normal morphologic appearance but could not be fertilized [8]. The low fertility rates of the oocytes can be caused by the perturbation of the division spindle of the second meiosis division. It is assumed that the disruption of the division spindle is caused by the osmotic stress. In cryoprotective solutions, because of the higher osmotic pressure, a dehydration of the cells takes place, which leads to disturbing the cell cytoskelet. Moreover, some studies showed that treating the oocytes with a hypotonic solution can induce the release of the cortical granules and a precocious hardening of the pellucid zone. The studies performed by Huang et. al. (2006) showed that the maxim osmotic tolerance of the oocytes exposed to a 2 mole L -1 solution of sucrose is 3 minutes, despite the fact that oocytes appear normal even after 20 minutes of exposure [7]. 4. Conclusions Maximum sucrose concentration in rehydration media should be 10%. Maximum concentration of trehalose in rehydration media should be 15%. 20% nonpenetrating cryoprotector in rehydration media results in loss of fecundate capacity of the oocyte. At 1.5 mol. L -1 none of the penetrating cryoprotectors tested reduced fecundation rates regardless of oocyte stage or time of exposure (can be used in controlled freezing protocol). At 3 mol. L -1 none of the penetrating cryoprotectors tested reduced fecundation rates at 3 minutes exposure regardless of oocyte stage (can be used for OPS or SSV vitrification). At 5 mol. L -1 only 1.2 popandiol did not affect the fecundation rates of the mature and immature oocytes (can be used in CS vitrification). 7 mol. L - 1 exposure to penetrating cryoprotector results in loss of fecundate capacity of the oocytes. Maximum concentration of penetrating cryoprotector in rehydration media should be 3 mol L -1. For 0.25 ml straws trehalose should be used as nonpenetrating cryoprotector. 102

6 Ficoll should not be used as nonpenetrating cryoprotector in oocyte freezing media. Acknowledgements This work was supported by PN II PCCA 120/2012. References 1. Kuwayama, M., Oocyte Cryopreservation, J. Mamm. Ova.Res., 2007, 24, 2-7; 2. Leibo, S. P., Cryopreservation of oocytes and embryos: Optimization by theoretical versus empirical analysis, Theriogenology, 2008, ; 3. Cean A., Bogdan A.T., Păcală N., Ivan A., Ilie D. E., Mammalian Oocyte Cryopreservation Review, Scientific Papers: Animal Science and Biotechnologies, 2011, 44 (1), p ; 4. Nagy A., Getsenstein M., Vintersten K. and Behringer R., Manipulating the mouse embryo: a laboratory manual third edition, Cold Spring Harbor Laboratory Press, New York, 2003 ISBN ; 5. Telea (Cean) A, Păcală N., Ivan Alexandra, Carabă V., Korbuly B., Studies regarding the crioprotective proprieties of the vitrification media, with glycerol, sucrose and ficoll 70 used in embryo cryopreservation, Scientific Papers: Animal Science and Biotechnologies, 2007, 40 (1), Kuleshova LL, Shaw JM, Trounson AO, Studies on replacing most of the penetrating cryoprotectant by polymers for embryo cryopreservation. Cryobiology Aug; 43(1): Huang Jack, Y. J., Hai-Ying Chen, Seang Lin Tan and Ri-Cheng Chian, Effects of Osmotic Stress and Cryoprotectant Toxicity on Mouse Oocyte Fertilization and Subsequent Embryonic Development In Vitro, Cell Preservation Technology, 2006, 4, Kuleshova, L. L., MacFarlane, D. R., Trounson, A.O., Sugars exert a major influence on the vitrification properties of ethylene glycol-based solutions and have low toxicity to embryos and oocytes. Cryobiology; 1999, 38,

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