Effect of different vitrification solutions and cryodevices on viability and subsequent development of buffalo oocytes vitrified at the germinal vesicle (GV) stage

The cryopreservation of immature oocytes would generate a readily available, non-seasonal source of female gametes for research and reproduction. In domestic animals, the most promising results on oocyte cryopreservation have been reported in cattle, few studies have been conducted on buffalo. The aim of the present study was to compare the use of different vitrification solutions and various cryodevices on viability and developmental competence of buffalo oocytes vitrified at the germinal vesicle (GV) stage. Cumulus oocyte-complexes (COCs) obtained at slaughterhouse from mature buffalo ovaries were randomly divided into three main groups and vitrified by using either straw or open pulled-straw (OPS) or solid surface vitrification (SSV) in a solution composed of either 20% ethylene glycol (EG) + 20% glycerol (GLY); VS1 or 20% EG + 20% dimethylsulfoxide (DMSO); VS2, respectively. Following vitrification and warming, viable COCs were matured in vitro for 22 h. Some COCs were denuded and stained with 1.0% aceto-orcein to evaluate nuclear maturation, whereas the others were fertilized and cultured in vitro for 7 days to determine the developmental competence. Although the recovery rate (64.9%) was the lowest in the oocytes vitrified by SSV using 20% EG + 20% DMSO as compared to the other groups, the best survival rate of the COCs was achieved in the same treatment (96.7%), which was significantly higher (P < 0.05) than those vitrified using traditional straws (71.8% in VS1 and 73.6% in VS2) or those vitrified using OPS and VS1 (73.9%). Furthermore, in the nuclear maturation test, the highest maturation rate (75.5%) was achieved in SSV vitrified COCs using 20% EG + 20% DMSO (VS2), which was similar to the controls (77.1%). Post IVF and embryo culture, the highest cleavage and blastocyst development rates were obtained in COCs vitrified in 20% EG + 20% DMSO using SSV (47.1% and 24.0%, respectively), which showed no difference from the controls (61.2% and 46.9%, respectively). Our results clearly show that the combination of SSV and 20% EG + 20% DMSO could be used effectively to vitrify GV stage buffalo COCs.     

Expression of CD9 and CD81 in bovine germinal vesicle oocytes after vitrification followed by in vitro maturation

The present study aimed to investigate the effect of vitrification on the expression of fertilization related genes (CD9 and CD81) and DNA methyl transferases (DNMT1 and DNMT3b) in bovine germinal vesicle (GV) oocytes and their resulting metaphase Ⅱ (MⅡ) stages after in vitro maturation culture. GV oocytes were vitrified using the open-pulled straw method; after warming, they were cultured in vitro. The vitrified-warmed GV oocytes and more developed MII oocytes were used to calculate the maturation rates (first polar body extrusion under a stereomicroscopy), and to detect mRNA expression (qRT-PCR). Fresh GV oocytes and their in vitro-derived MII oocytes served as controls. The results showed that both the maturation rate (54.23% vs. 42.93%) and the relative abundance of CD9 mRNA decreased significantly (p < 0.05) in bovine GV oocytes after vitrification, but the expression of CD81 and DNMT3b increased significantly. After in vitro maturation of vitrified GV oocytes, the resulting MII oocytes showed lower (p < 0.05) mRNA expression of genes (CD9, CD81, DNMT1 and DNMT3b) when compared to the control group (MII oocytes). Altogether, vitrification decreased the maturation rate of bovine GV oocytes and changed the expression of fertilization related genes and DNA methyl transferases during in vitro maturation.     

Comparison between the temperatures of intracellular ice formation in fresh mouse oocytes and embryos and those previously subjected to a vitrification procedure

When cells that have been subjected to supposedly innocuous freezing or vitrification procedures are used as the source material for subsequent experiments, it is important that they possess or exhibit the same relevant properties as fresh cells. In this study, we compared the temperatures of intracellular ice formation (IIF) in previously vitrified mouse oocytes/embryos with those in fresh intact ones. In the case of MII oocytes, 2-cell embryos, 4-6-cell embryos, and morulae, there are no significant differences (p > 0.05); namely, -33.3 °C (fresh) vs. -35.4 °C (vitrified) with MII oocytes, -40.6 °C (fresh) vs. -38.7 °C (vitrified) with 2-cell embryos, -38.0 °C (fresh) vs. -39.4 °C (vitrified) with 4-6-cell embryos, -24.5 °C (fresh) vs. -24.2 °C (vitrified) with morulae. But, in 8-cell embryos, there is a significant difference (p < 0.05) between fresh (−37.9 °C) and vitrified (−32.9 °C). If we include this significant difference, the overall IIF temperature of fresh cells is 0.74 °C lower than that of previously vitrified cells. If we exclude it, the IIF temperature for fresh cells is 0.32 °C higher than that for previously vitrified cells. Our conclusion then is that there is no difference between the IIF temperatures of fresh and previously vitrified cells.     

Melatonin rescues the aneuploidy in mice vitrified oocytes by regulating mitochondrial heat product

Vitrification of germinal vesicle (GV) stage oocytes has been shown to be closely associated with decreased rates of meiosis maturation and increased rates of aneuploidy. However, little is known about the effects of melatonin on these events in mice vitrified GV oocytes. In this study, the effects of melatonin on meiosis maturation potential and the incidence rate of aneuploidy in mouse vitrified oocytes were analyzed by supplementing in vitro maturation (IVM) solution with melatonin at different concentrations. This study, for the first time, showed that the mitochondrial heat production was markedly increased in vitrified oocytes (P < 0.05), which compromised the first polar body extrusion (PBE) of vitrified oocytes (73.3% vs. 85.1%, P < 0.05). However, 10⁻¹¹ mol/L melatonin could significantly decrease mitochondrial heat production and ROS level (9.1 vs. 12.0 pixels, P < 0.05), meanwhile increase ATP level (1.1 vs. 0.88 pmol, P < 0.05) and mtDNA copies (107438 vs. 67869, P < 0.05), which rescued the abnormal chromosome alignment (32% vs. 69%, P < 0.05) and reduced the incidence of aneuploidy (15.6% vs. 38.5%, P < 0.05) in vitrified oocytes. The meiosis maturation ability of vitrified oocytes with melatonin supplementation was similar to that of fresh ones (83.4% vs. 85.1%, P > 0.05). Collectively, our data revealed that melatonin has a protective action against vitrification-induced injuries of oocytes meiosis maturation.     

Effect of different cryopreservation protocols on cytoskeleton and gap junction mediated communication integrity in feline germinal vesicle stage oocytes

Oocyte cryopreservation in carnivores can significantly improve assisted reproductive technologies in animal breeding and preservation programs for endangered species. However, the cooling process severely affects the integrity and the survival of the oocyte after thawing and may irreversibly compromise its subsequent developmental capability.In the present study, two different methods of oocyte cryopreservation, slow freezing and vitrification, were evaluated in order to assess which of them proved more suitable for preserving the functional coupling with cumulus cells as well as nuclear and cytoplasmic competence after warming of immature feline oocytes.From a total of 422 cumulus enclosed oocytes (COCs) obtained from queens after ovariectomy, 137 were stored by vitrification in open pulled straws, 147 by slow freezing and 138 untreated oocytes were used as controls. Immediately after collection and then after warming, functional coupling was assessed by lucifer yellow injection and groups of fresh and cryopreserved oocytes were fixed to analyze tubulin and actin distribution, and chromatin organization. Finally, COCs cryopreserved with both treatments were matured in vitro after warming. In most cases, oocytes cryopreserved by slow freezing showed a cytoskeletal distribution similar to control oocytes, while the process of vitrification induced a loss of organization of cytoskeletal elements. The slow freezing protocol ensured a significantly higher percentage of COCs with functionally open and partially open communications (37.2 vs. 19.0) and higher maturational capability (32.5 vs. 14.1) compared to vitrified oocytes. We conclude that although both protocols impaired intercellular junctions, slow freezing represents a suitable method of GV stage cat oocytes banking since it more efficiently preserves the functional coupling with cumulus cells after thawing as well as nuclear and cytoplasmic competence. Further studies are needed to technically overcome the damage induced by the cryopreservation procedures on immature mammalian oocytes.     

Feasibility of a nylon-mesh holder for vitrification of bovine germinal vesicle oocytes in subsequent production of viable blastocysts

To improve the feasibility of nylon-mesh holder for vitrification of bovine cumulus-oocytes complexes (GV-COCs) having germinal vesicle, this study was conducted to demonstrate effects of sugars and protocol of exposure in vitrification on subsequent in vitro maturation, ultrastructural changes, and in vitro development in bovine immature oocytes after cryopreservation using nylon mesh. Before vitrification, GV-COCs were exposed to the cryoprotectant, which was composed of 40% (v/v) ethylene glycol, 18% (w/v) Ficoll-70, and 0.3 M sucrose (EFS40) or 0.3 M trehalose (EFT40), either by single step or in a stepwise way. The maturation rates in the stepwise exposure with EFS40 or EFT40 were significantly higher (P < 0.05) compared with the corresponding rates in the single step. In the stepwise exposure, few abnormalities were observed compared with the single-step exposure, where most oocytes showed a highly vacuolated cytoplasm with many ruptured mitochondria. Cleavage rates in fertilized oocytes previously exposed stepwise to EFS40 or EFT40 were significantly higher than those exposed by the single-step procedure. The cleaved embryos derived from the stepwise exposure to EFS40 developed to blastocysts. After transfer of blastocysts derived from vitrified GV oocytes, a female calf was born. These results indicate that vitrification of large numbers of bovine GV-COCs using a nylon-mesh holder accompanied with stepwise exposure minimizes structural damage in organelles, resulting in yield of viable blastocysts following in vitro embryo production.     

Effect of 6-dimethylaminopurine on germinal vesicle breakdown of bovine oocytes

The effect of 6-dimethylaminopurine (6-DMAP) on germinal vesicle breakdown (GVBD) and maturation in bovine oocytes was investigated in this study. This puromycin analog has been shown to be an inhibitor of phosphorylation. Whereas GVBD occurred in nearly all oocytes (96.8%, 120/124) in control medium, presence of 6-DMAP (2 mM) blocked this process almost completely, irrespective of the presence (98.3% GV, 349/355) or absence (97.1% GV, 165/170) of cumulus cells. When lower concentrations of 6-DMAP were used (100-500 microM), GVBD was observed in 87.9% of oocytes, but their maturation was arrested at late diakinesis-metaphase I stage. The inhibition of GVBD was fully reversible, but most of the metaphase II plates were abnormal (80%). To assess whether the action of 6-DMAP is different from the inhibitors of protein synthesis, metaphase II oocytes were exposed to either cycloheximide or 6-DMAP, respectively. Whereas in cycloheximide-supplemented medium approximately 80% of the oocytes were activated, parthenogenetic activation was much less frequent after incubation in 6-DMAP (14.5%). Fusion studies showed that, even if GVBD occurs in 6-DMAP supplemented medium, the level of the maturation-promoting factor (MPF) is decreased. These experiments may indicate the importance of phosphorylation for GVBD in cattle oocytes.     

Vitrification of murine mature metaphase II oocytes perturbs DNA methylation reprogramming during preimplantation embryo development

Accurate reprogramming of DNA methylation occurring in preimplantation embryos is critical for normal development of both fetus and placenta. Environmental stresses imposed on oocytes usually cause the abnormal DNA methylation reprogramming of early embryos. However, whether oocyte vitrification alters the reprogramming of DNA methylation (5 mC) and its derivatives in mouse preimplantation embryo development remains largely unknown. Here, we found that the rate of cleavage and blastocyst formation of embryos produced by IVF of vitrified matured oocytes was significantly lower than that in control counterparts, but the quality of blastocysts was not impaired by oocyte vitrification. Additionally, although vitrification neither altered the dynamic changes of 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5 fC) before 4-cell stage nor affected the levels of 5 mC and 5-carboxylcytosine (5caC) throughout the preimplantation development, vitrification significantly reduced the levels of 5hmC and 5 fC from 8-cell stage onwards. Correspondingly, vitrification did not alter the expression patterns of Tet3 in preimplantation embryos but apparently reduced the expression levels of Tet1 in 4-cell and 8-cell embryos and increased the expression levels of Tet2 at morula stage. Taken together, these results demonstrate that oocyte vitrification perturbs DNA methylation reprogramming in mouse preimplantation embryo development.     

Potential applications of sheep oocytes as affected by vitrification and in vitro aging

The present study was carried out to investigate how the interactions between aging, vitrification and post-warming interval affect the credibility of sheep MII-oocytes for in vitro fertilization (IVF), intracytoplasmic injection (ICSI), and parthenogenetic activation (PA). According to our results, aged oocytes had significantly higher rates of chromosome and spindle abnormalities compared to young oocytes. However after vitrification-warming, the total rates of these abnormalities were not significantly different between aged and young oocytes. Unvitrified-aged, and vitrified young and aged oocytes had comparable ultrastructural characteristics, whereas they were completely dissimilar in compared with unvitrified-young oocytes. Although mRNA abundance was reduced during vitrification-warming in both aged and young oocytes, the post-warming interval could improve the relative mRNA abundance. Aged oocytes had lower capacity for IVF and ICSI in compared with young oocytes, but had similar pattern for PA process. The vitrification process decreased developmental competence of both aged and young oocytes in compared with young ones, particularly when warmed oocytes were rested for 2 h before IVF, ICSI and PA. The results of the present study showed that in vitro aged oocytes had higher capacity to be used for parthenogenetic studies rather than IVF and ICSI. Furthermore, it was shown that vitrified oocytes had a time-dependent decline in quality and developmental potential. Notably, the speed of this decline was higher in vitrified-young oocytes, indicating that the vitrified oocytes do not require to be rested post warming. Conclusively, the results of this study can be useful in preserving in vitro aged oocytes to provide a valuable and easy access source of oocytes for research purposed studies.     

The effect of osmotic stress on the cell volume, metaphase II spindle and developmental potential of in vitro matured porcine oocytes

Porcine animal models are used to advance our understanding of human physiology. Current research is also directed at methods to produce transgenic pigs. Cryobanking gametes and embryos can facilitate the preservation of valuable genotypes, yet cryopreserving oocytes from pigs has proven very challenging. The current study was designed to understand the effects of anisotonic solutions on in vitro matured porcine oocytes as a first step toward designing improved cryopreservation procedures. We hypothesized that the proportion of oocytes demonstrating a normal spindle apparatus and in vitro developmental potential would be proportional to the solution osmolality. Oocytes were incubated for 10 min at 38 degrees C in various hypo- or hypertonic solutions, and an isotonic control solution and then assessed for these two parameters. Our results support the hypothesis, with an increasing proportion of spindles showing a disrupted structure as the levels of anisotonic exposure diverge from isotonic. Only about half of the oocytes maintained developmental potential after exposure to anisotonic solutions compared to untreated controls. Oocyte volume displayed a linear response to anisotonic solutions as expected, with an estimated relative osmotically inactive cell volume of 0.178. The results from this study provide initial biophysical data to characterize porcine oocytes. The results from future experiments designed to determine the membrane permeability to various cryoprotectants will allow predictive modeling of optimal cryopreservation parameters and provide a basis for designing improved cryopreservation procedures.     

Optimization of triacetate cellulose hollow fiber vitrification (HFV) method for cryopreservation of in vitro matured bovine oocytes

The aim of the current work was to evaluate applicability of triacetate cellulose hollow fiber vitrification (HFV) method for cryopreservation of groups of in vitro matured bovine oocytes (12–17 oocytes per device). We also attempted to optimize HFV protocol by altering concentration of non-permeating cryoprotectant (sucrose) in vitrification solution and concentration of extracellular Ca²⁺ by using a calcium-free base medium for preparation of vitrification/rewarming solutions with ethylene glycol (EG) as a single permeating cryoprotectant. Neither of modifications of HFV protocol significantly affected survival or fertilization rates of the vitrified bovine oocytes. Embryo development rates in the vitrification groups were lower than those in the control (31.2% of blastocysts at Day 8 post IVF). Use of vitrification/rewarming solutions with lower Ca²⁺ concentration and EG did not significantly improve embryo development rates. An increase of sucrose concentration in vitrification solution from 0.5 to 1.0 M significantly improved blastocyst yield on Day 8 post IVF (21.1–23.4% vs 3.1–3.5%; p < 0.05). Obtained results indicated that sufficient dehydration of the oocytes and/or the solution surrounding them in hollow fiber before immersion into liquid nitrogen is an important factor for successful vitrification. Use of HFV method allowed simplification and standardization of vitrification/rewarming procedures. Triacetate cellulose hollow fibers can be used successfully for cryopeservation of groups of in vitro matured bovine oocytes.     

Extrusion and removal of lipid from the cytoplasm of porcine oocytes at the germinal vesicle stage: centrifugation under hypertonic conditions influences vitrification

In the present study, we examined a novel lipid removal method, centrifugation in solutions made hypertonic by adding 0.27 M sugar. This allowed the lipid to be extruded and removed without the loss of active mitochondria or extra cytoplasm. The type of sugar influenced the proportion of oocytes that could be stratified by centrifugation. Glucose induced the highest extrusion rate of lipid droplets. After vitrification the rates of survival, germinal vesicle breakdown and metaphase II were 30, 26, and 7%, respectively, for lipid-removed GV oocytes; this was significantly higher (P<0.05) than for corresponding vitrified lipid-intact oocytes (2, 0, and 0%, respectively). These results indicated that this method is useful to remove whole lipid droplets without losing mitochondria and improves cryotolerance of porcine GV oocytes.     

Effects of trehalose co-incubation on in vitro matured prepubertal ovine oocyte vitrification

Our aim was to evaluate if loading prepubertal ovine oocyte with trehalose would impact on their further developmental potential in vitro and if it would improve their survival to vitrification procedures. COCs matured in vitro with (TRH) or without (CTR) 100mM trehalose were tested for developmental potential after in vitro fertilization and culture. Trehalose uptake was measured by the antrone spectrophotometric assay. No differences were recorded between the two experimental groups in fertilization rates (91.1 CTR vs 92.5% TRH), cleavage rates calculated on fertilized oocytes (96.1 CTR vs 95.4% TRH), first cleavage kinetic (56.1 CTR vs 51% TRH), and blastocyst rates (14.3 CTR vs 13.0% TRH). Anthrone assay revealed that in TRH group trehalose concentration/oocyte was 2.6microM. MII oocytes were then vitrified using cryoloops in TCM 199 containing 20% FCS, sucrose 0.5M, 16.5% Me(2)SO, 16.5% EG and plunged in LN(2). After warming, oocytes from TRH and CTR groups were tested for membrane integrity using the propidium iodide (PI)/Hoechst differential staining, and for developmental ability after in vitro fertilization. Trehalose in maturation medium affected membrane resistance (P<0.01) to vitrification/warming but not fertilization and cleavage rates. The differential staining showed a lower number of PI positive cells in TRH group compared to CTR one (14.3 vs 24.7%, respectively). Fertilization rates and cleavage rates did not differ between the two groups (55.3 and 41% for TRH and 47.7 and 41.7% for CTR, respectively). In conclusion trehalose in maturation medium stabilizes cell membranes during vitrification/warming of prepubertal ovine oocytes but does not affect fertilization and cleavage rates after warming.     

Aging changes the chromatin configuration and histone methylation of mouse oocytes at germinal vesicle stage

Aging decreases the fertility of mammalian females. In old oocytes at metaphase II stage (MII) there are alterations of the chromatin configuration and chromatin modifications such as histone acetylation. Recent data indicate that alterations of histone acetylation at MII initially arise at germinal vesicle stage (GV). Therefore, we hypothesized that the chromatin configuration and histone methylation could also change in old GV oocytes. In agreement with our hypothesis, young GV oocytes had non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) chromatin configurations, while old GV oocytes also had chromatin configurations that could not be classified as NSN or SN. Regarding histone methylation, young GV and MII oocytes showed dimethylation of lysines 4, 9, 36 and 79 in histone 3 (H3K4me2, H3K9me2, H3K36me2, H3K79me2), lysine 20 in histone H4 (H4K20me2) and trimethylation of lysine 9 in histone 3 (H3K9me3) while a significant percentage of old GV and MII oocytes lacked H3K9me3, H3K36me2, H3K79me2 and H4K20me2. The percentage of old oocytes lacking histone methylation was similar at GV and MII suggesting that alterations of histone methylation in old MII oocytes initially arise at GV. Besides, the expression of the histone methylation-related factors Cbx1 and Sirt1 was also found to change in old GV oocytes. In conclusion, our study reports changes of chromatin configuration and histone methylation in old GV oocytes, which could be very useful for further understanding of human infertility caused by aging.     

Production of good-quality porcine blastocysts by in vitro fertilization of follicular oocytes vitrified at the germinal vesicle stage

We investigated survival, meiotic competence, cytoplasmic maturation, in vitro fertilization, and development of immature porcine (Sus scrofa) oocytes cryopreserved by a modified solid surface vitrification protocol. Cumulus-oocyte complexes (COCs) collected from follicles 3 to 6 mm in diameter in abattoir-derived ovaries of prepubertal gilts were either vitrified (Vitrified group), subjected to cryoprotectant treatment (CPA group), or used without any treatment (Control group). Oocyte viability was assayed by staining with fluorescein diacetate. Live oocytes were matured in vitro and their meiotic progression investigated by nuclear staining. In a series of experiments, the glutathione (GSH) content of in vitro-matured oocytes and viability of cumulus cells were assayed simultaneously. The in vitro-matured oocytes were also fertilized and cultured in vitro to assess their ability to be fertilized and to develop to the blastocyst stage, respectively. The proportion of viable oocytes in the Vitrified group was significantly lower than that in the CPA and Control groups (27.7%, 90.4%, and 100%, respectively). Among the three groups, there were no differences in meiotic competence, cumulus viability, and GSH levels at the end of in vitro maturation. Fertilization parameters (i.e., rates of male pronucleus formation, monospermy, and second polar body extrusion) were also similar among groups. However, comparison of the developmental abilities of oocytes in the Vitrified, CPA, and Control groups revealed that the Vitrified group had a significantly reduced ability to undergo first cleavage (34.4%, 63.3%, and 69.0%) and to develop to the blastocyst stage (5.1%, 25.5%, and 34.6%). The mean total cell numbers in blastocysts after 6 d of culture were not significantly different among the Vitrified, CPA, and Control groups (40.3, 42.8, and 43.4). In conclusion, despite low survival rates and impaired development in the Vitrified group, meiotic competence, cytoplasmic maturation, and subsequent fertilization characteristics of surviving germinal vesicle oocytes were unaffected by vitrification, and high-quality blastocysts were produced from vitrified immature oocytes.     

Maturation of the reconstructed oocytes by germinal vesicle transfer in rabbits and mice

The present study was designed to evaluate the feasibility of germinal vesicle (GV) transfer in rabbits and mice. The GV oocytes were collected from ovaries and cultured in 20 microg/mL 3-isobutyl-1-methylxanthin (IBMX) in TCM199 medium, which caused oocytes to shrink, enlarging the perivitelline space to facilitate the GV removal and transfer. Pairs of GV-cytoplast complexes were fused with electric pulses, and the fused, reconstructed oocytes were cultured in TCM199 for 24 h. Results are as follows: 1) The exposure time of rabbit GV oocytes to IBMX medium affected the success of GV removal. For oocytes cultured for 2 and 3 h in IBMX medium, removed rates were 56% and 44, respectively, significantly higher (P < 0.05) than removal rates of GV oocytes cultured for 1 and 4 h (27% and 27%, respectively); 2) There was no significant difference (P > 0.1) in fusion and maturation rates of rabbit reconstructed oocytes collected at 72 and 84 h after initiation of FSH injection to donors; 3) eCG in the maturation media improved development of rabbit-to-rabbit GV transferred oocytes but had no positive effect on mouse-to-rabbit GV transferred oocytes; 4) When mouse GV-karyoplasts were injected into enucleated rabbit oocytes, fusion rates of GV-karyoplasts measuring 40- to 50-microm and 80- to 90-microm in diameters obtained were 84% and 93%, respectively. The rates were significantly higher (P < 0.05) than fusion rates after transferring GV-karyoplasts measuring 30- to 35-microm in diameter (63%). The maturation rate (89%) of reconstructed oocytes composed of 80- to 90-microm mouse GV-karyoplasts and rabbit GV-enucleated cytoplasts was higher than that seen for oocytes composed of 40- to 50-microm (77%, P<0.05) or 30- to 35-microm (59%, P<0.01) mouse karyoplasts. Thirty-five of the 63 (56%) mature mouse-to-rabbit reconstructed oocytes had the normal complement of 20 chromosomes.     

Temperature-dependent osmotic behavior of germinal vesicle and metaphase II stage bovine oocytes in the presence of Me2SO in relationship to cryobiology

Plasma membrane permeability coefficients and their activation energies (Ea) for water (Lp) and dimethyl sulfoxide (PMe2SO) as well as the reflection coefficient (sigma) were determined for germinal vesicle (GV) and metaphase II (MII) bovine oocytes. A micropipette perfusion technique was used with a temperature controlled circulation chamber, which was adapted to a micromanipulator. Experiments were performed at five different temperatures (30, 20, 10, 4 and -3 degrees C). The Kedem and Katchalsky model was assumed and L(p), P(Me2SO) and sigma were estimated. Estimated permeability values from the experimental temperatures were then applied to Arrhenius plots In(Lp) or In(PMe2SO) vs 1/Temperature (K) to estimate the activation energies (Ea) for L(p)Me2SO and P(Me2SO). The estimated E(a) for L(p)Me2SO for GV and MII oocytes were 23.84 Kcal/mol and 8.46 Kcal/mol, respectively. The E(a) for P(Me2SO) were 21.0 Kcal/mol and 23.20 Kcal/mol, respectively. The correlation (r2) for these linear regression plots for GV oocytes were 0.83 and 0.95 for L(p)Me2SO and P(Me2SO), respectively. For MII oocytes, r2 values were 0.95 and 0.99 for L(p)Me2SO and P(Me2SO), respectively. There was a possible discontinuity detected in the Arrhenius plot for L(p)Me2SO for GV oocytes. A significant decrease of the reflection coefficient was observed at 10 degrees C compared to other experimental temperatures. These data provide a fundamental basis that should be taken into account for low temperature preservation of bovine oocytes in the presence of Me2SO.     

Effects of cryodevice type and donors' sexual maturity on vitrification of minke whale (Balaenoptera bonaerensis) oocytes at germinal vesicle stage

Germinal-vesicle-stage oocytes enclosed with compact cumulus cell layers (COCs) were recovered from adult or prepubertal minke whale ovaries, and were vitrified in a solution containing 15% ethylene glycol, 15% DMSO and 0.5 M sucrose using either a Cryotop or an open-pulled straw (OPS) as the cryodevice. The post-warm COCs with normal morphology were cultured for 40 h in a 390 mosmol in vitro maturation medium, and oocytes extruding the first polar body were considered to be matured. The proportion of morphologically normal COCs after vitrification and warming was higher when the COCs were cryopreserved by Cryotop (adult origin, 88.4%; prepubertal origin, 80.8%) compared with the OPS (adult origin, 67.7%; prepubertal origin, 64.2%). The oocyte maturation rate was higher in the adult/Cryotop group (29.1%) compared with those of the prepubertal/Cryotop group (14.4%), the adult/OPS group (14.3%) and the prepubertal/OPS group (10.6%). These results indicate that the Cryotop is a better device than the OPS for vitrification of immature oocytes from adult minke whales.     

Effect of oocyte vitrification on DNA damage in metaphase II oocytes and the resulting preimplantation embryos

As an assisted reproduction technology, vitrification has been widely used for oocyte and embryo cryopreservation. Many studies have indicated that vitrification affects ultrastructure, gene expression, and epigenetic status. However, it is still controversial whether oocyte vitrification could induce DNA damage in metaphase II (MII) oocytes and the resulting early embryos. This study determined whether mouse oocytes vitrification induce DNA damage in MII oocytes and the resulting preimplantation embryos, and causes for vitrification‐induced DNA damage. The effects of oocyte vitrification on reactive oxygen species (ROS) levels, γ‐H2AX accumulation, apoptosis, early embryonic development, and the expression of DNA damage‐related genes in early embryos derived by in vitro fertilization were examined. The results indicated that vitrification significantly increased the number of γ‐H2AX foci in zygotes and two‐cell embryos. Trp53bp1 was upregulated in zygotes, two‐cell embryos and four‐cell embryos in the vitrified group, and Brca1 was increased in two‐cell embryos after vitrification. Vitrification also increased the ROS levels in MII oocytes, zygotes, and two‐cell embryos and the apoptotic rate in blastocysts. Resveratrol (3,5,4′‐trihydroxystilbene) treatment decreased the ROS levels and the accumulation of γ‐H2AX foci in zygotes and two‐cell embryos and the apoptotic rate in blastocysts after vitrification. Overall, vitrification‐induced abnormal ROS generation, γ‐H2AX accumulation, an increase in the apoptotic rate and the disruption of early embryonic development. Resveratrol treatment could decrease ROS levels, γ‐H2AX accumulation, and the apoptotic rate and improve early embryonic development. Vitrification‐associated γ‐H2AX accumulation is at least partially due to abnormal ROS generation.