The influence of cooling rate and warming rate on the response of renal cortical slices frozen to −40 °C in the presence of 2.1 M cryoprotectant (ethylene glycol, glycerol, or dimethyl sulfoxide)

Abstract To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to achieve this is to convert the water inside the cells into a non-crystalline glass. It is currently believed that to accomplish this vitrification, the cells must be suspended in a very high concentration (20–40%) of a glass-inducing solute, and subsequently cooled very rapidly. Herein, we report that this belief is erroneous with respect to the vitrification of one-cell rat embryos. In the present study, one-cell rat embryos were vitrified with 5 μL of EFS10 (a mixture of 10% ethylene glycol, 27% Ficoll, and 0.45 M sucrose) in cryotubes at a moderate cooling rate, and warmed at various rates. Survival was assessed according to the ability of the cells to develop into blastocysts and to develop to term. When embryos were vitrified at a 2,613 °C/min cooling rate and thawed by adding 1 mL of sucrose solution (0.3 M, 50 °C) at a warming rate of 18,467 °C/min, 58.1 ± 3.5% of the EFS10-vitrified embryos developed into blastocysts, and 50.0 ± 4.7% developed to term. These rates were similar to those of non-treated intact embryos. Using a conventional cryotube, we achieved developmental capabilities in one-cell rat embryos by rapid warming that were comparable to those of intact embryos, even using low concentrations (10%) of cell-permeating cryoprotectant and at low cooling rates.

Download Full-text

cis-5-Cyclooctene-1,2-dione and its Ethylene Ketals

Canadian Journal of Chemistry ◽

10.1139/v72-246 ◽

1972 ◽

Vol 50 (10) ◽

pp. 1548-1556 ◽

Cited By ~ 18

Author(s):

Peter Yates ◽

E. G. Lewars ◽

P. H. McCabe

Keyword(s):

Hydrogen Peroxide ◽

Acetic Anhydride ◽

Ethylene Glycol ◽

Dimethyl Sulfoxide

Oxidation of cis-cis-1,5-cyclooctadiene with hydrogen peroxide gives cis-5-cyclooctene-trans-1,2-diol (3) which is converted to cis-5-cyclooctene-1,2-dione (6) on treatment with dimethyl sulfoxide and acetic anhydride. Bromination of 6 is accompanied by transannular bonding to give a dibromo keto ether 9a or b. Ketalization of 6 with ethylene glycol gives a monoketal 11 and two diketals 12 and 13 with 1,3-dioxolane and 1,4-dioxane rings, respectively. Bromination of 12 with bromine or pyridinium perbromide is accompanied by transannular bonding and fission of one of the 1,3-dioxolane rings to give a dibromo monoketal ether 15a (or b). Bromination of 12 with N-bromosuccinimide followed by dehydrobromination gives a cyclooctadiene-1,2-dione diketal 20a (or b).

Download Full-text

Stimulatory and Inhibitory Effects of Dimethyl Sulfoxide and Ethylene Glycol on ATPase Activity and Calcium Transport of Sarcoplasmic Membranes

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1977.tb11430.x ◽

1977 ◽

Vol 74 (3) ◽

pp. 611-621 ◽

Cited By ~ 47

Author(s):

Rudy THE ◽

Wilhelm HASSELBACH

Keyword(s):

Ethylene Glycol ◽

Atpase Activity ◽

Dimethyl Sulfoxide ◽

Calcium Transport ◽

Inhibitory Effects

Download Full-text

64 CRYOPRESERVATION OF CAT OVARIAN TISSUE BY VITRIFICATION

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab64 ◽

2013 ◽

Vol 25 (1) ◽

pp. 179 ◽

Cited By ~ 1

Author(s):

J. Galiguis ◽

C. E. Pope ◽

M. C. Gómez ◽

C. Dumas ◽

S. P. Leibo

Keyword(s):

Ethylene Glycol ◽

Dimethyl Sulfoxide ◽

Ovarian Tissue ◽

Genetic Material ◽

Cortical Tissue ◽

Rock Inhibitor ◽

Tissue Samples ◽

Preantral Follicles ◽

Wide Range ◽

Chi Square Analysis

The cryopreservation of ovarian tissue is linked to a wide range of possible applications, from oocyte harvesting to allo- and xenotransplantation. These procedures have significant potential for the preservation of valuable genetic material and endangered-species conservation. The objectives of the present study were to (1) compare viability of preantral follicles obtained from fresh v. vitrified feline ovarian cortex, (2) evaluate the effect of apoptotic inhibitors (ROCK inhibitor v. glutathione) on viability of follicles from vitrified samples, and (3) determine the optimal inhibitor concentration for follicle viability. In Experiment 1, 5 × 5 × 1 mm cortical tissue samples were obtained from excised cat ovaries and assigned to either the fresh control or vitrification group. Fresh samples were processed through a 230-micron-pore dissection strainer to collect preantral follicles. Follicles were then stained in Trypan blue to determine membrane integrity and survival rates. Vitrification samples were first equilibrated in 7.5% dimethyl sulfoxide and 7.5% ethylene glycol at ~22°C and then in vitrification solution consisting of 20% dimethyl sulfoxide, 20% ethylene glycol, and 0.5 M sucrose. They were then vitrified on a thin, perforated, metal strip (Cryotissue, Kitazato Biopharma, Fujinomiya, Japan). Samples were later warmed in 1.0 M sucrose at 38°C. Follicles were then collected and assessed for survival. In Experiment 2, follicles were collected from samples vitrified/warmed in cryo-media supplemented with either 3 × 104 nM ROCK inhibitor or 6 nM glutathione. Follicles from samples vitrified/warmed without inhibitor treatment were used as controls. In Experiment 3, tissue samples were vitrified/warmed in cryo-media supplemented with 0, 2, 6, or 10 nM glutathione before follicle viability was determined. Data were evaluated by chi square analysis. In Experiment 1, 637 and 340 follicles were collected from fresh and vitrified samples, respectively. Overall, survival was higher in freshly collected follicles when compared to those from the vitrified group (67 v. 18%, respectively; P < 0.05). Evaluation of apoptotic inhibitors was determined through collection of 314, 354, and 506 follicles from inhibitor-free, ROCK inhibitor, and glutathione-treated media, respectively. Follicles from samples vitrified in inhibitor-free media and in ROCK inhibitor survived at a lower rate than those from glutathione-treated samples (10 and 13% v. 18%, respectively; P < 0.05). In Experiment 3, a total of 539, 641, 625, and 632 follicles were collected from samples treated in 0, 2, 6, and 10 nM glutathione, respectively. There were no statistical differences in follicle survival among the 0, 2, and 6 nM groups. However, follicles treated in 10 nM glutathione survived at a higher rate than those vitrified/warmed in the absence of glutathione (20 v. 14%; P < 0.05). In summary, viability of preantral follicles from ovarian cortical tissue was significantly reduced by vitrification. Despite this, tolerance of such follicles to cryopreservation was improved by vitrifying and warming in cryo-media containing 10 nM glutathione. Partially funded by the LSU/ACRES Collaborative Project.

Download Full-text

38 Vitrification of prepubertal lamb spermatogonia using a novel vitrification system

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab38 ◽

2019 ◽

Vol 31 (1) ◽

pp. 145 ◽

Cited By ~ 1

Author(s):

S. Ledda ◽

S. Pinna ◽

S. Nieddu ◽

D. Natan ◽

A. Arav ◽

...

Keyword(s):

Ethylene Glycol ◽

In Vitro Culture ◽

Dimethyl Sulfoxide ◽

Fertility Preservation ◽

Testicular Tissue ◽

Gonadal Tissue ◽

New Device ◽

Vitrification Solution ◽

Tissue Cryopreservation

Vitrification is a method extensively used for preserving oocytes and embryos and is also gaining acceptance for preserving gonadal tissue. Cryopreservation of spermatogonial stem cells is an applicable method for young males seeking fertility preservation before starting a treatment or can be a tool for genetic preservation of rare or high-value animals. The aim of this work was to evaluate the cryopreservation of testicular tissue from young lambs by vitrification using a new device named E.Vit (FertileSafe, Ness Ziona, Israel) that permits all cryopreservation procedures to be performed in straw. The new device consists of a 0.3-mL straw (Cryo Bio System, IMV, L’Aigle, France) with a capsule containing 50-µm pores inserted at one end. Testicular tissue extracts were prepared from testes of slaughtered lambs (n=10, 40 days old), opened by sagittal sectioning with a microblade and collecting small pieces of testicular tissue (1mm3) from the middle part of the rete testis. Three pieces of gonadal tissue were inserted into each E.Vit device. Each straw was sequentially loaded vertically in two 1.5-mL microtubes, which contained the following solutions: first, the equilibrating solution (7.5% dimethyl sulfoxide+7.5% ethylene glycol+20% FCS in TCM-199) for 6min, followed by 90min in the vitrification solution (18% dimethyl sulfoxide+18% ethylene glycol+0.5M Trehalose+BSA in TCM-199). After exposure to the equilibrating solution and vitrification solution, the solutions were removed and the straws were directly loaded into LN2. The warming procedure consisted of placing the straws directly into 5-mL tubes containing 100, 50, and 25% warming solution (1M sucrose in TCM-199+20% FCS) at 38.6°C for 5min each before arrival into the holding medium. Samples were recovered from the straws incubated at 38.6°C in 5% CO2 in air in TCM 199+5% FCS and evaluated at 0 and 2h post-warming for viability using trypan blue staining. Expression of a panel of specific genes (SOD2, HSP90b, BAX, POUF5/OCT4, TERT, CIRBP, KIF11, AR, FSHR) was analysed by real-time PCR in cryopreserved tissue in vitro cultured for 2h post-warming (2hV), in fresh controls immediately after tissue dissection (0hF), and after 2h of in vitro culture (2hF). The majority of cells survived after vitrification, although viability immediately after warming (0hV: 56%±1.45) or after 2h of in vitro culture (IVC) (2hV: 54±7%) was significantly lower compared with non-cryopreserved fresh controls (0hF: 89%±1.45; ANOVA P<0.05). Expression analysis showed specific patterns for the different genes. Notably, BAX transcript abundance was not affected by vitrification or IVC, indicating an acceptable level of stress for the cells. The genes HSP90b and CIRBP were down-regulated in 2hF but increased in 2hV, as expected. Expression of SOD1 and OCT4 was altered by vitrification but not by IVC. Conversely, expression of TERT, KIF11, and AR was affected by both IVC and cryopreservation (ANOVA P<0.05). This novel protocol for testicular tissue cryopreservation of prepubertal animals may be a promising strategy for fertility preservation and can contribute as a new approach in the development of large-scale biodiversity programs.

Download Full-text

25 Effect of taxol and epothilone B on meiotic spindle stabilization in vitrified bovine oocytes

Reproduction Fertility and Development ◽

10.1071/rdv33n2ab25 ◽

2021 ◽

Vol 33 (2) ◽

pp. 120

Author(s):

E. Girka ◽

K. R. Bondioli

Keyword(s):

Ethylene Glycol ◽

Dimethyl Sulfoxide ◽

Recovery Period ◽

Normal Chromosome ◽

Meiotic Spindle ◽

Control Group ◽

Chromosome Arrangement ◽

Epothilone B ◽

Significant Difference ◽

Bovine Oocytes

Vitrification has the potential to be a valuable technique for preservation of bovine oocytes; however, this method often results in abnormal microtubule and chromosome arrangement. The aim of this experiment was to evaluate taxol and epothilone B as meiotic spindle stabilising pretreatments in a vitrification protocol. Bovine oocytes were purchased and matured invitro during shipment. At 18h of maturation, oocytes were divided randomly into control, taxol, and epothilone B treatments (Table 1). All treatments were prepared in invitro maturation (IVM) medium (IVF Biosciences). Partially denuded oocytes were incubated in either control or treatment medium for 15min at 38.5°C before vitrification. Oocytes were incubated in an equilibration solution (10% dimethyl sulfoxide, 10% ethylene glycol) for 5min, transferred to a vitrification solution (20% dimethyl sulfoxide, 20% ethylene glycol, 0.5M sucrose), loaded onto a Cryolock, and plunged into liquid nitrogen within 45s. For warming, a Cryolock was placed directly into a 0.5M sucrose solution and incubated for 3min. Oocytes were transferred to a 0.25M solution for 3min and washed in the basal solution used for vitrification and warming media (Dulbecco’s phosphate-buffered saline, 20% fetal bovine serum). Once warmed, oocytes were transferred to IVM medium for a 4-h recovery period and completely denuded before staining. Staining to evaluate spindle morphology was performed with anti α-tubulin primary antibody and secondary antibody Alexa Fluor 488. Oocytes were also stained with Hoechst to evaluate chromosome arrangement. Both spindle morphology and chromosome arrangement data were analysed using a logistic regression with a binomial response variable (normal/abnormal). Both 0.5μM and 1.0μM Taxol treatments had no effect on either meiotic spindle or chromosome arrangement compared with the control group (P>0.05). The 2.0μM taxol treatment improved chromosome configuration (P<0.05) with no effect on microtubule distribution compared with the control group (P>0.05). All epothilone B treatments resulted in disruption of microtubule distribution and chromosome arrangement compared with control (P<0.001) and resulted in a consistent abnormality hypothesised to be tubulin polymerization. These results indicate that taxol is capable of increasing the occurrence of normal chromosome arrangement in vitrified bovine oocytes and that epothilone B may cause additional harm to the oocyte that is not associated with the metaphase plate. Table 1. Effect of stabilisation agents on meiotic spindle of invitro-matured bovine oocytes Treatment n Normal microtubule distribution (%) Normal chromosome arrangement (%) Control 100 44 47 0.5μM Taxol 104 44 37 1.0μM Taxol 98 43 56 2.0μM Taxol 102 49 62a 0.5μM Epothilone B 103 11b 11b 1.0μM Epothilone B 97 6b 8b 2.0μM Epothilone B 100 2b 1b aP<0.05;. bP<0.001: Different superscripts within a column indicate a significant difference.

Download Full-text

28 Effect of deuterium oxide on bovine oocyte cryotolerance

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab28 ◽

2019 ◽

Vol 31 (1) ◽

pp. 140

Author(s):

F. Salerno ◽

M. Rubessa ◽

B. Gasparrini ◽

M. Wheeler

Keyword(s):

Ethylene Glycol ◽

Dimethyl Sulfoxide ◽

Bovine Serum ◽

In Vitro Maturation ◽

Deuterium Oxide ◽

Control Group ◽

Crystal Formation ◽

Cleavage Rate ◽

Maturation Medium

It is known that cryopreservation triggers spindle disassembly, increased aneuploidy risk, decreased post-thaw survival, fertilization, and embryo development. We hypothesised that a treatment with D2O before vitrification would slow down oocyte metabolism and reduce ice crystal formation by replacing water inside the cells. The aim of the study was to evaluate the effect of a 4-h treatment with different D2O concentrations (0, 3, 15, and 30%) on cryotolerance of bovine in vitro-matured oocytes. Abattoir-derived bovine oocytes were matured in vitro for 20h in TCM-199 medium with 15% of bovine serum (BS), 0.5µg mL−1 of FSH, 5µg mL−1 of LH, 0.8mM l-glutamine, and 50µg mL−1 of gentamicin at 39°C with 5% of CO2 and randomly divided into 5 experimental groups. A group of non-vitrified oocytes was used as the fresh oocyte control group, whereas the remaining oocytes were incubated for 4h in in vitro maturation medium with 0% (vitrified control; n=205), 3% (n=205), 15% (n=205), and 30% D2O (n=205) before vitrification. The experiment was repeated 4 times. Oocytes were denuded in HEPES-buffered TCM-199 (H199)+5% BS and vitrified using a cryotop freezing straw. The oocytes were incubated in 200μL of H199+20% BS with 7.5% ethylene glycol and 7.5% dimethyl sulfoxide for 3min. After that, oocytes were collected in 50μL of H199+20% fetal bovine serum with 15% ethylene glycol+15% dimethyl sulfoxide and 0.5M sucrose for 20s and plunged into LN2. One month later, oocytes were warmed in thawing media with decreasing concentrations of sucrose (1.35M to 0.31M) and then placed into in vitro maturation medium for 2h before IVF. Matured oocytes were IVF and cultured according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-1355). Cleavage and blastocyst rates were evaluated after 7 days of culture. Data were analysed using the GLM procedure of SPSS (SPSS Inc., Chicago, IL, USA). The least statistical difference post-hoc test was used to perform statistical multiple comparison. The α-level was set at 0.05. As expected, both cleavage [60.5±4.6 (fresh control); 36.9±2.6 (0% D2O); 46.3±3.7 (3% D2O); 31.6±2.4 (15% D2O); and 24.4±2.6 (30% D2O)] and blastocyst rates [25.7±0.8 (fresh control); 9.0±0.8 (0% D2O); 9.0±0.7 (3% D2O); 3.6±0.2 (15% D2O); and 4.3±0.8 (30% D2O)] decreased in all vitrified groups compared with the fresh control group. Within vitrified oocytes, cleavage rate increased (P<0.05) with 3% D2O treatment compared with the other groups. However, pretreatment with higher (15-30%) D2O concentrations decreased (P<0.05) blastocyst rates of vitrified-warmed oocytes. In conclusion, a pretreatment with low concentrations (3%) of D2O improved the cleavage rate of bovine vitrified-warmed oocytes, suggesting a potential beneficial effect, whereas deleterious effects were observed using the higher concentrations. Therefore, further studies are required to assess a potential use of D2O to improve oocyte cryotolerance, likely testing different incubation times.

Download Full-text

44 EFFECT OF CRYOPROTECTANT AGENTS IN EQUINE OVARIAN BIOPSY FRAGMENTS

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab44 ◽

2016 ◽

Vol 28 (2) ◽

pp. 152

Author(s):

G. D. A. Gastal ◽

B. G. Alves ◽

S. O. Paiva ◽

K. A. Alves ◽

S. G. S. de Tarso ◽

...

Keyword(s):

Ethylene Glycol ◽

Dimethyl Sulfoxide ◽

Propylene Glycol ◽

Cell Density ◽

Stromal Cell ◽

Ovarian Tissue ◽

Rank Correlation ◽

Analysis Data ◽

Exact Test ◽

Spearman’S Rank Correlation

Studies on ovarian tissue preservation in horses are scarce, and only one research group (Devireddy et al. 2006 Mol. Reprod. Develop. 73, 330–341) has tested the effect of the cryoprotectant agents in equine ovarian tissue. The purpose of this study was to evaluate (1) the effect of cryoprotectant agents in the morphology of equine preantral follicles (PAF) and structure of the ovarian tissue, (2) the relationship between ovarian stromal cell density and PAF density, and (3) the association of ovarian stromal cell density with PAF morphology after exposure to cryoprotectant agents. Three independent experiments with identical methodologies were performed in this study. Each experiment was composed by one cryoprotectant agent (dimethyl sulfoxide, ethylene glycol, propylene glycol) performed in 3 replicates. Ovarian biopsy fragments (1.5 × 1.5 × 10 mm) were harvested from 6 mares and submitted to 4 times of exposure (0, 10, 15, 20 min) at room temperature in a solution of α-minimal essential medium (MEM)+ and one cryoprotectant (dimethyl sulfoxide, ethylene glycol, or propylene glycol) at a concentration of 1.5 M. The cryoprotectant removal was performed by a 3-step (5 min each) equilibration process using (1) α-MEM + 0.5 M sucrose, (2) α-MEM + 0.25 M sucrose, and (3) α-MEM alone after each time of exposure. All fragments were fixed in Bouin’s solution for histological analysis. Data were analysed by ANOVA, Fisher’s exact test, Spearman’s rank correlation, and power test. The PAF density, stromal cell density, and area of the fragments were not affected (P > 0.05) by any of the cryoprotectant agents throughout the time of exposure. However, the morphology of the PAF was affected (P < 0.05) by the cryoprotectant agents. In the propylene glycol and dimethyl sulfoxide, higher (P < 0.05) percentages of abnormal PAF were observed at 10 and 20 min of exposure, respectively. However, the PAF morphology in the ethylene glycol treatments was not affected (P > 0.05) throughout the times of exposure. Strong correlations (range, 0.57 to 0.77; power, 96 to 99%) were identified between PAF density and stromal cell density in all experiments. When stromal cells were classified into groups of density (i.e. low, 25.67–34.66; medium, 34.67–43.66; and high, 43.67–52.67 cells/2500 μm2), only the high stromal cell density was positively correlated (P < 0.001) with the PAF density; however, no correlation between stromal cell density and PAF morphology was observed. In conclusion, (1) ethylene glycol seems to be a less harmful cryoprotectant agent to equine PAF, (2) exposure to cryoprotectant agents did not affect the cell density and area of ovarian fragments, (3) PAF density was positively correlated with stromal cell density, and (4) stromal cell density did not affect the morphology of PAF.

Download Full-text