High survival of mouse oocytes using an optimized vitrification protocol

The method of vitrification has been widely used for cryopreservation. However, the effectiveness of this method for mammalian oocytes could be improved by optimizing each step of the process. In the present study, we tested the effects of varying several key parameters to determine the most effective protocol for mouse oocyte vitrification. We found that cryoprotectant containing ethylene glycol and dimethylsulfoxide plus 20% fetal calf serum produced the highest rates of oocyte survival, fertilization, and blastocyst formation. The duration and temperature of oocyte exposure to vitrification and thawing solutions influenced survival rate. The presence of cumulus cells surrounding oocytes and the incubation of thawed oocytes in Toyoda-Yokoyama-Hosoki medium also increased oocyte survival. Open pulled straw and nylon loop methods were more effective than the mini-drop method. Finally, the combination of these improved methods resulted in better spindle morphology when compared to the unimproved methods. These results demonstrate that the outcomes of mouse oocyte vitrification can be improved by a suitable combination of cryopreservation methods, which could be applied to future clinical research with human oocytes.

Mature OCCs were equilibrated in VS1 for 2 mins and VS2 for 20 secs prior to incubation in TS1 (M199 + 20% FCS + 0.33 mol/l sucrose) for 3 mins and TS2 (M199 + 20% FCS + 0.25 mol/l sucrose) for 3 mins. For the control group, OCCs were incubated in HM followed by incubation in TS1 and TS2. All procedures were conducted at room temperature. After three washes in TYH medium, all oocytes were subjected to insemination and embryo culture.
Effect of temperature of exposure to VS and TS on oocyte survival, fertilization, and blastocyst formation. Next, we tested the effect of temperature of exposure to VS and TS on OCC vitrification. OCCs were exposed to VS at 25 °C and TS at 25 °C, VS at 25 °C and TS at 37 °C, VS at 37 °C and TS at 25 °C, or VS at 37 °C and TS at 37 °C. All other procedures were same as those previously described. We found no differences between the two groups exposed to VS at 25 °C (survival: 74.6 ± 2.0% and 72.1 ± 1.0%; fertilization: 53.4 ± 1.2% and 50.6 ± 2.8%; blastocyst formation: 42.6 ± 1.8% and 41.6 ± 2.3%, respectively; Fig. 5A-C). Similarly, there were no differences between the two groups exposed to VS at 37 °C (survival: 36.8 ± 2.3% and 31.8 ± 0.9%; fertilization: 19.8 ± 0.9% and 18.5 ± 0.9%; blastocyst formation: 11.1 ± 0.6% and 8.3 ± 0.5%, respectively). However, groups exposed to VS at 25 °C showed greater oocyte survival, fertilization, and blastocyst formation than groups exposed to VS at 37 °C. Thus, OCCs were exposed to VS and TS at 25 °C in the following experiments.
Combinations of optimized key parameters improved oocyte spindle morphology. Procedures of freezing and thawing can damage oocyte spindle, which may affect oocyte survival, fertilization, and embryo development. In our study, spindles of oocytes from improved and unimproved vitrification combinations were  compared. In the improved group, the best conditions at each steps were used for oocytes cryopreservation; whereas in the unimproved group, the worst conditions at each steps were used for oocytes cryopreservation (Fig. 9A). After thawing followed by 3 hours incubation in CZB medium, most of the spindles reappeared (Fig. 9B). Our results showed that improved group have higher percent of normal spindle than that in the unimproved group (70.6 ± 8.8% and 32.4 ± 2.1%, p < 0.05, Fig. 9C).

Discussion
Cryoprotectant is the most important component of successful vitrification. High concentrations of cryoprotectant inside cells become hyaloid material under ultra-cooling conditions, which prevents the formation of ice crystals that are harmful to cells. However, a high concentration of cryoprotectant has toxic effects that may lead to oocyte damage. Thus, both permeability and toxicity need to be considered when determining the optimal concentration of cryoprotectant. Many types of cryoprotectants, such as Me2SO, PrOH, and EG, have been used for oocyte cryopreservation [9][10][11] . A previous study reports that EG has the lowest toxicity 12 , whereas Me2SO causes ZP hardening and prevents fertilization 13,14 . Some studies suggest that using a mixed reagent containing different cryoprotectants exerts less toxicity 15 . In the present study, we observed high rates of survival, fertilization, and embryo development when cryoprotectant consisted of EG and Me2SO. Furthermore, this combination of cryoprotectant was less toxic to oocytes than each cryoprotectant used separately. Although GLY and PrOH are often used as cryoprotectants, their permeabilities are not as good as those of EG and Me2SO. Because a high concentration of cryoprotectant does not allow long periods of equilibration in VS, the permeation of GLY and PrOH into cells is insufficient. Therefore, a combination of GLY and PrOH is not suitable for oocyte vitrification.
Although a higher concentration of cryoprotectant results in better dehydration of cells, the ability of oocytes to endure toxicity should also be considered. In other words, finding a balance between high cryoprotectant concentration and low toxicity is key to successful vitrification. We found that a total of concentration of 40% was somewhat toxic to oocytes, but a total concentration of 20% did not lessen the toxic effects on oocyte survival and embryo development. Thus, a concentration of 40% appears to be optimal for successful vitrification. A recent study in pigs shows that cryoprotectant composed of 15% Me2SO and 15% EG improved the in vivo development of vitrified mature oocytes 16 . However, the amount of lipids and the size of oocytes differ substantially between mice and pigs, which may explain differences between species in the optimal concentration of cryoprotectant.
After vitrification, oocytes exhibit poor capacity for fertilization due to ZP hardening. Previous studies report that oocytes vitrified in VS containing FCS show a comparable fertilization rate as control oocytes without VS treatment [17][18][19] . This is because the existence of FCS in the perivitelline space extrudes residual cryoprotectant, resulting in increased fertilization. Also, FCS has been found to prevent ZP hardening through its fetuin content 20 . We found that the removal of FCS from VS worsened the outcomes of oocyte vitrification. Also, our results suggest that using 10% FCS is probably not sufficient to prevent ZP hardening. Surprisingly, 20% FCS was associated with the highest rate of oocyte survival, fertilization, and blastocyst formation, and 30% FCS did not produce better results. Therefore, higher concentrations of FCS do not appear to benefit the vitrification process. Another reason responsible for this is that high FCS can prevent cryoprotectant from permeating into oocytes, and then leads to the formation of ice crystals and damage oocytes.
The reduction of equilibration time in VS is an effective way to reduce the damage caused by cryoprotectant 21 . However, larger oocytes require longer exposure to VS for full dehydration. To resolve this conflict, we employed a two-step equilibration protocol, which allows sufficient time for oocytes to dehydrate but also minimizes the harm of high VS concentration. We found that an equilibration time of 2 mins in VS1 and 20 or 30 secs in VS2 resulted in the greatest survival, fertilization, and blastocyst formation, whereas less time in VS (i.e., 1 min in VS and 10 secs in VS2) may not be sufficient to permeate oocytes. However, our results also suggest that longer exposure to cryoprotectant may do more harm than good.
The process of oocyte thawing involves an increase in temperature, exclusion of cryoprotectant inside cells, and rehydration. Incorrect thawing methods can lead to reformation of ice crystals and damage to the subcellular structure of oocytes. In this context, osmotic pressure is a key factor affecting vitrification outcomes. Including sucrose in the TS provides a higher osmotic pressure that minimizes the rapid movement of water 21 . Also, a sufficient length of time is necessary for substituting TS for VS. We obtained the best results by employing two steps of VS removal and found that an incubation time of less than 3 mins in TS was not sufficient to completely remove VS, which had a negative effect on oocyte survival and embryo development.
The temperature at which oocytes are exposed to VS and TS also affects vitrification outcomes. Higher temperatures are associated with greater toxic effects of cryoprotectant due to sudden changes in osmotic pressure. Lower temperature can reduce the toxicity of cryoprotectants 22 . We found that a VS temperature of 25 °C allowed cryoprotectant to infiltrate oocytes quickly with less toxicity. However, when the temperature was increased to 37 °C, oocytes could not endure the faster change in osmotic pressure, resulting in lower survival and compromised developmental potential. As for the temperature of TS, both 25 °C and 37 °C resulted in good rates of survival and embryo development. However, a recent study reported that human oocytes rehydrated at 37 °C get higher survival than those rehydrated at room temperature 23 . Inconformity of results may due to the differences of cryoprotectants, freeing and warming time, especially species. Moreover, except choosing a suitable temperature for freezing and warming, the cooling and warming rates maybe important. Recently, a study on human oocyte reported that they have determined freezing and warming rates using a data logger. A novel, aseptic closed system vitrification technique for the cryopreservation of embryos and oocytes has been developed and clinically validated in there study 24 .
Although a previous study reports that cumulus cells do not contribute to oocyte survival after freezing 25 , accumulating data suggest that cumulus cells and their extracellular matrix promote fertilization 26,27 . We found that oocytes surrounded by fully intact or partial cumulus layers had a higher fertilization rate than denuded oocytes, which is consistent with previous results 28 . Based on these findings, we conclude that the presence of cumulus layers promotes oocyte survival after freezing. We hypothesize that VS-induced changes in osmotic pressure inside oocytes can be minimized by the presence of cumulus cells. The smaller size of cumulus cells compared with oocytes may enable cumulus cells to tolerate high concentrations of cryoprotectant, although this possibility needs further investigation.
Cytoskeletal structure is very sensitive to temperature changes. A study by Eroglu et al. showed that incubation of oocytes in fertilization medium for 1 hour after freezing rescues cytoskeletal disruption, which benefits fertilization and embryo development 29 . In our study, oocytes incubated in TYH for 15 or 30 mins showed increased fertilization and embryo development, although this step did not promote oocyte survival. We propose that recovery in fertilization medium for a short time before fertilization allows the reorganization of tubulin and microfilament components inside oocytes, which in turn leads to better fertilization and embryo development. Similar to our results, some studies in human reported that spindles recovered for a period of time after vitrification improve the embryo development 30,31 and this recovery may make the vitrified oocyte has comparable cleavage timings, cell number, and DNA methylation patterns with the freshed oocytes 32 .
A key step of vitrification is the rapid decrease in temperature of tissue. In the present study, we compared three types of previously described carriers for oocyte vitrification 16,33,34 . We found no differences among carriers in terms of fertilization and embryo development. However, OPS and nylon loop methods resulted in greater survival than the mini-drop method. OPS and nylon loop methods were performed by quickly placing oocytes into liquid nitrogen, whereas the mini-drop method was performed by dropping liquid onto the surface of iron at a temperature of − 196 °C. Thus, the greater survival of oocytes frozen with OPS and nylon loop methods may be due to the faster decrease in temperature. In addition, using the mini-drop method, it was difficult to recover oocytes from the storage tank, making this method not very applicable for oocyte cryopreservation.
In summary, our results demonstrate that the composition and concentration of cryoprotectant, duration and temperature of exposure to VS, TS, and TYH, presence of cumulus cells, and type of carrier affects the outcomes of mouse oocyte vitrification. The best outcomes can be obtained by using an optimized vitrification protocol.

Materials and Methods
Ethics statement. All  Oocyte freezing and thawing. Oocytes subjected to cryopreservation were transferred to holding medium (HM; TCM199 + 20% (v/v) fetal calf serum (FCS)) prior to sequential equilibration in vitrification solution 1 (VS1) and VS2. The gentle mixture of oocytes with VS occurred immediately after transfer to ensure adequate equilibrium. After equilibration in VS2, oocytes were loaded into one of three types of carriers: open pulled straw (OPS; Fig. 1, path A), nylon loop ( Fig. 1; path B), or mini-drop ( Fig. 1; path C). OPSs were made in-house by polishing commercial plastic tubes (Tianshankaifeng Company, Beijing, China) into thin-walled tubes (outer diameter = 200 μ m, inner diameter = 180 μ m) with fire. Nylon loops were made by fixing fine nylon onto iron wires. Mini-drop was carried out by dropping liquid at a volume of less than 20 μ l onto an iron surface immersed in liquid nitrogen.
For experiments without freezing, oocytes were transferred from VS2 directly to thawing solution 1 (TS1; Fig. 1, path D). Oocytes subjected to freezing were rapidly placed into liquid nitrogen with their carriers. After 1 week of storage in liquid nitrogen, carriers were collected and immediately transferred to TS1. After incubation with TS1, oocytes were observed under an inverted microscope, transferred to TS2, and then placed in HM. Temperature control during exposure to VS or TS was conducted using MSP Selectable Temperature (Thermo Plate, Japan). Thawed oocytes were then transferred from HM to Toyoda Yokoyama Hosoki (TYH) medium (Fig. 1).
In vitro fertilization and embryo culture. Adult (12-14 weeks of age) male B6D2 F1 mice were used for sperm collection. Sperm suspension collected from the epididymis was held for 2 hours in 200 μ l TYH medium supplemented with 4 mg/ml bovine serum albumin (BSA). Recovered MII oocytes were incubated with spermatozoa for 6 hours in 200 μ l TYH medium supplemented with 4 mg/ml BSA. The final sperm concentration for fertilization was 1 × 10 6 /ml. Zygotes were cultured in Chatot-Ziomet-Bavister (CZB) medium without glucose in a humidified atmosphere of 5% CO 2 at 37 °C for the first 2 days and then transferred to CZB medium supplemented with 5.5 mmol/l glucose when embryos reached the four-cell stage. Embryo development was observed 24 and 96 hours after fertilization to calculate the percentage of fertilization and blastocyst formation, respectively.
Immunofluorescence and Confocal Microscopy. After thawing, oocytes were incubated in CZB medium for 3 hours to allow reformation of spindle. Oocytes were then fixed with 4% paraformaldehyde for 40 mins and then permeabilized with 0.5% Triton X-100 for 2.5 hours. Following blocking in 1% BSA in PBS containing 1/1,000 Tween-20 and 1/10,000 Triton X-100 for 1 hour. Then samples were incubated overnight at 4 °C with mouse anti-TUBB antibody (Abcam). Chromosomes were stained with DAPI (5μ g/ml, Roche, Mannheim, Germany) for 10 mins. After staining, samples were mounted on glass slides using vectashield (Vector Labs, Burlingame, CA) mounting medium and examined with a confocal laser scanning microscope (Nikon, A1R, Japan).
Statistical analysis. The total number of oocytes for each group was at least 400. Data are presented as mean + SEM (n = 3). Differences among groups in survival (number of surviving oocytes/number of recovered oocytes), fertilization (number of two-cell zygotes/number of surviving oocytes), and blastocyst formation (number of blastocysts/number of surviving oocytes) was evaluated using χ 2 tests. A p-value < 0.05 was considered statistically significant.