Cryopreservation Of Mouse Oocytes Research Articles

The effect of solution nonideality on modeling transmembrane water transport and diffusion-limited intracellular ice formation during cryopreservation.

A new model was developed to predict transmembrane water transport and diffusion-limited ice formation in cells during freezing without the ideal-solution assumption that has been used in previous models. The model was applied to predict cell dehydration and intracellular ice formation (IIF) during cryopreservation of mouse oocytes and bovine carotid artery endothelial cells in aqueous sodium chloride (NaCl) solution with glycerol as the cryoprotectant or cryoprotective agent. A comparison of the predictions between the present model and the previously reported models indicated that the ideal-solution assumption results in under-prediction of the amount of intracellular ice at slow cooling rates (<50 K/min). In addition, the lower critical cooling rates for IIF that is lethal to cells predicted by the present model were much lower than those estimated with the ideal-solution assumption. This study represents the first investigation on how accounting for solution nonideality in modeling water transport across the cell membrane could affect the prediction of diffusion-limited ice formation in biological cells during freezing. Future studies are warranted to look at other assumptions alongside nonideality to further develop the model as a useful tool for optimizing the protocol of cell cryopreservation for practical applications.

126 Establishment of a live young mouse production system using unfertilized oocytes cryopreserved via a simple vitrification method

Since the first successful reports into oocyte freezing, many papers concerning the cryopreservation of mouse oocytes have been published. However, a simple and practical cryopreservation method for unfertilized C57BL/6 mouse oocytes, and an IVF system using these cryopreserved oocytes have yet to be established, in spite of the fact that C57BL/6 is the prevalent inbred strain and is used for large-scale knockout programs. In this study, unfertilized C57BL/6 mouse oocytes were cryopreserved via a simple vitrification method. After warming, IVF was performed using cryopreserved unfertilized oocytes and fresh sperm, cryopreserved unfertilized oocytes and cold-stored sperm, cryopreserved unfertilized oocytes and frozen sperm (C57BL/6 strain sperm), and cryopreserved unfertilized oocytes and frozen sperm derived from GEM strains (C57BL/6 background GEM strains). Nearly all of the cryopreserved oocytes were recovered, of which over 90% were morphologically normal. Those oocytes were then used for in vitro fertilization, resulting in 72–97 % of oocytes developing into 2-cell embryos. A portion of the 2-cell embryos were transferred to recipients, resulting in live young being produced from 32% to 49 % of the embryos. Source of funding: This study was supported by a Grant-in-aid for National BioResource Project (NBRP) Fundamental Technologies Upgrading Program. No additional external funding was received for this study. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Conflict of interest: None declared. nakagata@gpo.kumamoto-u.ac.jp

Applications of cryopreserved unfertilized mouse oocytes for in vitro fertilization

Since the first successful reports into oocyte freezing, many papers concerning the cryopreservation of mouse oocytes have been published. However, a simple and practical cryopreservation method for unfertilized C57BL/6 mouse oocytes, and an IVF system using these cryopreserved oocytes have yet to be established, in spite of the fact that C57BL/6 is the prevalent inbred strain and is used for large-scale knockout programs. In this study, unfertilized C57BL/6 mouse oocytes were cryopreserved via a simple vitrification method. After warming, IVF was performed using cryopreserved unfertilized oocytes and fresh sperm, cryopreserved unfertilized oocytes and cold-stored sperm, cryopreserved unfertilized oocytes and frozen sperm (C57BL/6 strain sperm), and cryopreserved unfertilized oocytes and frozen sperm derived from GEM strains (C57BL/6 background GEM strains). Nearly all of the cryopreserved oocytes were recovered, of which over 90% were morphologically normal. Those oocytes were then used for in vitro fertilization, resulting in 72–97% of oocytes developing into 2-cell embryos. A portion of the 2-cell embryos were transferred to recipients, resulting in live young being produced from 32–49% of the embryos. In summary, we established the simple and practical method of mouse oocyte vitrification with high survivability and developmental ability and the IVF using the vitrified-warmed oocytes with fresh, cold-stored or cryopreserved sperm with high fertility.

Fertility potential of mouse oocytes revealed by cytosolic calcium after cryopreservation and in vitro maturation

OBJECTIVE: To test whether cryopreservation of mouse oocytes at an intermediate stage of maturation in vitro is optimal for developmental competence. DESIGN: Germinal vesicle (GV) oocytes were matured in vitro to metaphase II (MII) with or without cryopreservation at different stages. At MII they were activated by intracytoplasmic sperm injection (ICSI) and intracellular calcium oscillations were monitored to predict developmental competence. MATERIALS AND METHODS: 6 week-old B6D2F1 mice were primed with PMSG and oocytes harvested for cryopreservation in 1.5M 1,2-propanediol at GV, pro-metaphase I (GVBD) or MII stages, or used as fresh controls. Oocytes were matured to the MII stage after thawing, and ICSI was performed with B6D2F1 sperm using a Piezo drill. Calcium oscillations were subsequently measured in oocytes loaded with Fura-2/AM using a fluorescence microscope and InCyt 2 software to measure fluorescence ratios at 340/380 nm and determine intracellular calcium. The data were analyzed by non-parametric statistical tests and ANOVA. RESULTS: There were no significant differences in survival between the three stages at cryopreservation (84-94%). Fresh oocytes exhibited normal calcium spikes every 15-20 min post-fertilization lasting 3-4 hr. After cryopreservation at GV or GVBD stages, maturation to MII and ICSI, persistent calcium oscillations were induced similar to fresh oocytes, but they were truncated or absent in oocytes cryopreserved at the MII stage. CONCLUSIONS: Cryopreservation of oocytes prior to the MII stage appeared to improve developmental potential as indicated by their ability to support normal intracellular calcium oscillations. There was no reduction of survival after thawing. Since calcium signaling is essential for successful fertilization and an excellent indicator of competence, there may be an unexpected advantage of banking oocytes at GV or GVBD stages instead of MII, perhaps from a longer cellular recovery time after the stress of cryopreservation.

Protecting female fertility from cancer therapy

Recent advances in cancer therapy have improved the long-term survival of young cancer patients who are then commonly faced with iatrogenic infertility and premature ovarian failure. Preservation of fertility potential has thus become a major goal and could be realized by preventing ovarian toxicity or by cryopreservation of reproductive cells (i.e. oocytes, embryos) and tissues (i.e. ovarian cortex). GnRH analogs prevent chemotherapy-induced-ovarian-damage in rats, however human results are controversial. Anti-apoptotic agents (i.e. sphingosine-1-phosphate) may present an innovative treatment to prevent oocyte destruction during cancer therapy. Although cryopreservation of mouse oocytes is successful, the results obtained in other mammalian species were worse, probably due to their extreme sensitivity to suboptimal conditions during the process of cryopreservation. This resulted in low oocyte survival and fertilization rates, a high incidence of polyploidy, and poor embryonic developmental ability. Ovarian tissue cryopreservation is currently considered as the optimal procedure for follicle banking. Transplantation offers the best prospect of using frozen-thawed ovarian tissue, since no reliable ovarian in-vitro culture technology exists.

Vitrification of mouse oocytes using a nylon loop.

Cryopreservation of mouse oocytes was improved by the use of ultra-rapid vitrification using a nylon loop of 0.5 mm diameter. Oocytes that were vitrified using the loop survived at high rates and were fertilized following a small hole being made in the zona pellucida (69.8%) and developed to the blastocyst stage in culture (67.4%) at similar rates to that of oocytes that were not cryopreserved. Blastocysts resulting from oocytes vitrified using the nylon loop had similar development of the inner cell mass and trophectoderm as blastocysts from non-cryopreserved oocytes. In contrast, oocytes that were cryopreserved using a slow-freezing protocol where most of the Na+ is replaced with choline had lower rates of fertilization (39.5%), reduced development to the blastocyst stage (25.7%), and blastocysts had reduced development of the inner cell mass. Blastocysts derived from oocytes that were vitrified with the nylon loop were able to implant (88.0%) and develop into fetuses (56.5%) at significantly higher rates compared to blastocysts derived from oocytes that were slow-frozen (52.4 and 26.2%, respectively). Vitrification of mouse oocytes using the nylon loop results in the retention of viability of the oocytes and subsequent embryos.

Cryopreservation of Mouse Oocytes Using a Medium with Low Sodium Content: Effect of Plunge Temperature

The effect of various combinations of plunge temperature and thawing protocol on the survival and viability of mouse oocytes was examined. The oocytes were frozen either in a standard freezing medium (ETFM, embryo transfer freezing medium) or in a low-sodium, choline-based freezing medium (CJ2), with 1.5 M 1,2-propanediol and 0.1 M sucrose, and using a conventional slow cooling method. The criteria used to assess survival were morphological state after thawing (intact or lysed), ability to become fertilized, and ability to develop to the two-cell, morula, and blastocyst stage in vitro. Oocytes frozen in CJ2 and plunged into liquid nitrogen (LN2) from −10, −20, or −33°C remained intact and developed to the blastocyst stage at significantly higher rates than oocytes frozen in ETFM. For oocytes plunged into LN2 from −33°C, very rapid thawing (10 s in 30°C water) was more detrimental than rapid or slow thawing (holding in air at room temperature for 10 or 30 s, respectively, prior to submersion in water at 30°C for 10 s). By contrast, oocytes plunged into LN2 from −10 or −20°C survived better when thawing was very rapid or rapid than when thawing was slow. With the current protocol CJ2 was very effective over a wide range of plunge temperatures (−20 to −33°C), although the optimal thawing protocol depended on the particular plunge temperature. Over 90% of oocytes surviving after slow cooling in CJ2 to −33°C could be plunged to −196°C with little or no further damage.

Cytoskeleton and polyploidy after maturation and fertilization of cryopreserved germinal vesicle-stage mouse oocytes.

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. The GV oocytes were cryopreserved using a slow-cooling (0.5 degree C/min) and slow-thawing (8 degrees C/min) protocol in 1.5 M dimethylsulfoxide supplemented with 0.2 M sucrose and analyzed before and during fertilization by multiple-label fluorescence and differential interference contrast microscopy techniques. When examined after in vitro maturation, the vast majority (> 95%) of cryopreserved and control oocytes displayed normal microfilament and microtubule organization. With respect to barrel-shaped 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). 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.

Effects of cooling and equilibration in DMSO, and cryopreservation of mouse oocytes, on the rates of in vitro fertilization, development, and chromosomal abnormalities.

In a previous study, we have shown that the cryopreservation of mouse oocytes caused increases in the rates of degeneration and of digynic polyploid embryos, while the fertility of frozen-thawed oocytes was decreased. In this study, we have attempted to determine the different stages in the complete freezing-thawing process which are deleterious for the oocytes and the subsequent zygotes. IVF assays showed that DMSO decreased the fertility of oocytes, whereas cooling to 0 degrees C had no effect. DMSO, used at 0 degrees C, was less deleterious for oocytes. Thus, the prefreezing manipulations seem to be important for the quality and fertility of oocytes. However, neither DMSO nor cooling increased the incidence of chromosomal abnormalities in embryos obtained from inseminated exposed oocytes. Therefore, the increased frequency of polyploidy observed in embryos after the cryopreservation of mouse oocytes must correspond to disruption occurring during the freezing-thawing process.

Cryopreservation of Mouse Oocytes: Mutagenic Effects in the Embryo?1

We have shown in previous studies that the complete cycle of cryopreservation and prefreezing manipulations increases the degeneration and decreases the fecundability of mouse oocytes. The present study confirms these results. Moreover, we show that the increase of polyploidy previously observed in one-cell zygotes derived from frozen-thawed oocytes persists during the early stages of embryonic development. Furthermore, embryos obtained from frozen oocytes or oocytes exposed to prefreezing manipulations show an increase in the frequency of sister chromatid exchanges. Since the estimation of sister chromatid exchange is a sensitive test of mutagenicity, this suggests that the complete cycle of cryopreservation might alter the oocyte and, more particularly, induce DNA damage.