微流控芯片用于卵母细胞冷冻保存的实验研究

低温保存对卵母细胞造成渗透损伤、毒性损伤和冰晶损伤,使得细胞冻后质量难以提高.本文首次提出将微流控法添加-去除保护剂分别与三种冷冻载体(OPS、QC及Cryotop)搭配使用,对猪卵母细胞进行冷冻保存,并与传统冷冻法进行比较;然后,首次选用透明陶瓷和玻璃制作集成一体化芯片,对猪卵母细胞进行冷冻保存,以冷冻保存后的细胞存活率和发育率为判断依据,筛选出较好的方案;最后,对冻后卵母细胞的早期凋亡情况、胞内活性氧水平和线粒体膜电位水平进行分析.结果表明,微流控添加-去除保护剂组卵母细胞冻后存活率以及卵裂率都显著高于传统冷冻组,可以有效降低卵母细胞的早期凋亡率和胞内活性氧水平,减小线粒体损伤,提高细胞的冻后质量.透明陶瓷一体化芯片保存卵母细胞得到的存活率和卵裂率与传统OPS冷冻的保存结果无显著差异.微流控芯片技术为卵母细胞的低温保存提供新的思路,有较好的应用前景.     

Changes to the meiotic spindle and zona pellucida of mature mouse oocytes following different cryopreservation methods

This study is to investigate the change of morphology of the meiotic spindle and the extent of zona hardening relating to the morphological survival and developmental competence of thawed oocytes. Four- to 8-week-old female mice (C57BL/6) primed with an intraperitoneal injection of pregnant mare's serum gonadotropin and human chorionic gonadotropin. Cryopreserved oocytes using two protocols: vitrificaton using ethylene glycol (EG) and slow freezing using propanediol (PROH). The freezing oocytes were thawed and were fertilized and subsequently cultured in vitro. Spindle/chromosome imagery, dissolution of zona pellucida, and post-thawing survival and development were comparable between two groups. The vitrification cryopreservation method proved to be better than the slow-freezing protocol when comparing the frequency of normal-shaped spindle development post-thawing. The difference in the time required for the dissolution of the zona pellucida under treatment of pronase that was determined to exist between the two cryopreservation methods was statistically significant (P<0.005). The survival rate of post-thawed mature oocytes was significantly greater for the vitrification group than it was for the slow-freezing cryopreservation group (P=0.005). The vitrification cryopreservation of mature murine oocytes would appear to be more satisfactory than the slow controlled-rate freezing method as regards the post-thawing oocyte survival and also the incidence of the normal spindle apparatus in the ooplasm.     

Reversible disassembly of the actin cytoskeleton improves the survival rate and developmental competence of cryopreserved mouse oocytes

Effective cryopreservation of oocytes is critically needed in many areas of human reproductive medicine and basic science, such as stem cell research. Currently, oocyte cryopreservation has a low success rate. The goal of this study was to understand the mechanisms associated with oocyte cryopreservation through biophysical means using a mouse model. Specifically, we experimentally investigated the biomechanical properties of the ooplasm prior and after cryopreservation as well as the consequences of reversible dismantling of the F-actin network in mouse oocytes prior to freezing. The study was complemented with the evaluation of post-thaw developmental competence of oocytes after in vitro fertilization. Our results show that the freezing-thawing process markedly alters the physiological viscoelastic properties of the actin cytoskeleton. The reversible depolymerization of the F-actin network prior to freezing preserves normal ooplasm viscoelastic properties, results in high post-thaw survival and significantly improves developmental competence. These findings provide new information on the biophysical characteristics of mammalian oocytes, identify a pathophysiological mechanism underlying cryodamage and suggest a novel cryopreservation method.     

Quantitative investigations on the effects of exposure durations to the combined cryoprotective agents on mouse oocyte vitrification procedures

Vitrification by using two-step exposures to combined cryoprotective agents (CPAs) has become one of the most common methods for oocyte cryopreservation. By quantitatively examining the status of oocytes during CPA additions and dilutions, we can analyze the degree of the associated osmotic damages. The osmotic responses of mouse MII oocyte in the presence of the combined CPAs (ethylene glycol, EG, and dimethyl sulfoxide, DMSO) were recorded and analyzed. A two-parameter model was used in the curve-fitting calculation to determine the values of hydraulic conductivity (L(p)) and permeability (P(s)) to the combined CPAs at 25°C and 37°C. The effects of exposure durations and the exposure temperatures on the cryopreservation in terms of frozen-thawed cell survival rates and subsequent development were examined in a series of cryopreservation experiments. Mouse MII oocytes were exposed to pretreatment solution (PTS) and vitrification solution (VS) at specific temperatures. The PTS used in our experiment was 10% EG and 10% DMSO dissolved in modified PBS (mPBS), and the VS was EDFS30 (15% EG, 15% DMSO, 3 × 10(-3) M Ficoll, and 0.35 M sucrose in mPBS).The accumulative osmotic damage (AOD) and intracellular CPA concentrations were calculated under the different cryopreservation conditions, and for the first time, the quantitative interactions between survival rates, subsequent development rates, and values of AOD were investigated.     

Cryopreservation of zebrafish (Danio rerio) oocytes using improved controlled slow cooling protocols

Cryopreservation of gametes provides a promising method to preserve fish genetic material. Previously we reported some preliminary results on cryopreservation of zebrafish (Danio rerio) oocytes using controlled slow cooling and determined the optimum cryoprotective medium and cooling rate for stage III zebrafish oocytes. In the present study, the effects of two different cryopreservation media, cryoprotectant removal method, final sample freezing temperature before LN₂ plunge, warming rate, and the post-thaw incubation time on oocyte viability were investigated. Commonly used cryoprotectant methanol and glucose were used in this study. Stage III zebrafish oocytes were frozen in standard culture medium 50% L-15 or in a sodium-free KCl buffer medium. Oocyte viability was assessed using trypan blue staining and ATP assay. The viability of oocytes frozen in KCl buffer was significantly higher than oocytes frozen in L-15 medium. The results also showed that fast thawing and stepwise removal of cryoprotectant improved oocyte survival significantly, with highest viability of 88.0 ± 1.7% being obtained immediately after rapid thawing when assessed by trypan blue staining. However, after 2 h incubation at 22 °C the viability of freeze-thawed oocytes decreased to 29.5 ± 5.1%. Results also showed that the ATP level in oocytes decreased significantly immediately after thawing. All oocytes became translucent after freezing which complicated the use of GVBD test (in vitro maturation of oocytes followed by observation of germinal vesicle breakdown which results in oocytes becoming translucent). New oocyte viability assessment methods are urgently needed.     

Effects of cryoprotectant addition and washout methods on the viability of precision-cut liver slices

Successful vitrification of organ slices is hampered by both osmotic stress and chemical toxicity of cryoprotective agents (CPAs). In the present study, we focused on the effect of osmotic stress on the viability of precision-cut liver slices (PCLS) by comparing different CPA solutions and different methods of loading and unloading the slices with the CPAs. For this purpose, we developed a gradient method to load and unload CPAs with the intention of minimizing sudden changes in osmolarity and thereby avoiding osmotic stress in the slices in comparison with the commonly used step-wise loading/unloading approach. With this gradient method, the CPA solution was introduced at a constant rate into a specially designed mixing chamber containing the slices. We showed that immediate mixing of the infused CPA and the chamber constituents occurred, which enabled us to control the CPA concentration to which PCLS were exposed as a function of time.     

Cryopreservation of starfish oocytes

Research from many laboratories over the past several decades indicates that invertebrate oocytes and eggs are extraordinarily difficult to freeze. Since starfish oocytes, eggs, and embryos are an important cell and developmental biology model system, there is great interest to cryopreserve these cells. Previous starfish oocyte cryopreservation studies using slow cooling protocols revealed that these cells are highly sensitive to osmotic stress and form intracellular ice at very high sub-zero temperatures, suggesting that common freezing methodologies may not prove useful. We report here that a short exposure to 1.5 M Me2SO/1 M trehalose in hypotonic salt solution followed by ultra-rapid cooling to cryogenic temperatures allows starfish oocytes to be cryopreserved with the average survival rate of 34% when normalized to control oocytes that were exposed to CPA, but not frozen. On average, 51% of the oocytes in 77% of the batches of frozen oocytes underwent meiotic maturation in response to the starfish maturation hormone, 1-methyladenine. In one experiment, eggs developing from thawed oocytes were capable of being fertilized and two developed into embryos. These data suggests that successful cryopreservation of starfish oocytes is possible, but will need further refinement to increase the numbers of fully competent embryos.     

An integrated microfluidic device for single cell trapping and osmotic behavior investigation of mouse oocytes

Transport properties of oocytes play an important role in the optimization of their cryopreservation. However, there are still no systematical investigations on oocyte transport properties from the viewpoint of single-cell trapping and high precision perfusion, especially with the powerful microfluidic approach. To this end, we developed an easy-to-fabricate and easy-to-use microfluidic chip along with automatic single cell trapping capability to investigate the oocyte membrane transport properties. The experimental results indicate that the device is available and reliable. We further performed a comparative study of the oocyte membrane transport properties between single and multi-step CPA addition protocols and confirmed that the transport property parameters measured by single-step osmotic shift could not be used for prediction of the osmotic responses of oocytes in multi-step CPA addition. This study provides a powerful tool for investigation of oocyte osmotic responses.     

Effects of cryoprotectant addition and washout methods on the viability of precision-cut liver slices

Successful vitrification of organ slices is hampered by both osmotic stress and chemical toxicity of cryoprotective agents (CPAs). In the present study, we focused on the effect of osmotic stress on the viability of precision-cut liver slices (PCLS) by comparing different CPA solutions and different methods of loading and unloading the slices with the CPAs. For this purpose, we developed a gradient method to load and unload CPAs with the intention of minimizing sudden changes in osmolarity and thereby avoiding osmotic stress in the slices in comparison with the commonly used step-wise loading/unloading approach. With this gradient method, the CPA solution was introduced at a constant rate into a specially designed mixing chamber containing the slices. We showed that immediate mixing of the infused CPA and the chamber constituents occurred, which enabled us to control the CPA concentration to which PCLS were exposed as a function of time.With this method, CPA concentration versus time profiles were varied using various commercially available CPA mixtures [VMP, VM3, M22, and modified M22 (mM22)]. The viability of PCLS was determined after CPA loading and unloading and subsequent incubation during 3 h at 37 °C. Despite the reduction of osmotic stress, the viability of slices did not improve with gradual loading and unloading and remained considerably lower than that of untreated slices. The toxicity of the three CPA solutions did not correlate with either their potential osmotic effects or their total concentrations, and did not change strongly with exposure time in 100% CPA. The most likely explanation for these observations is that PCLS are not very sensitive to osmotic changes of the magnitude imposed in our study, and chemical toxicity of the CPA solutions is the main barrier to be overcome. The chemical toxicity of the CPAs used in this study probably originates from a source other than the total concentration of the solutions. The presented gradient method using the specially designed chamber is more time and cost effective than the step-wise method and can be universally applied to efficiently evaluate different CPA solutions.     

Cryopreservation of porcine embryos derived from in vitro-matured oocytes

This study describes a cryopreservation method for porcine in vitro-produced (IVP) embryos using as a model parthenogenetic embryos derived from in vitro-matured (IVM) oocytes. IVP embryos at the expanded blastocyst stage were cryopreserved by vitrification using the minimum volume cooling (MVC) method and exhibited an embryo survival rate of 41.2%. Survival was then significantly improved (83.3%, P < 0.05) by decreasing the amount of cytoplasmic lipid droplets (delipation) prior to vitrification. IVP embryos at the 4-cell stage also survived cryopreservation when vitrified after delipation (survival rate, 36.0%), whereas post-thaw survival of nondelipated embryos was quite low (9.7%). Furthermore, it was demonstrated that porcine IVP morulae can be cryopreserved by vitrification following delipation by a noninvasive method (survival rate, 82.5%). These results clearly confirm that porcine embryos derived from IVM oocytes can be effectively cryopreserved with high embryo survival using the MVC method in conjunction with delipation.     

Effect of maturation stage at cryopreservation on post-thaw cytoskeleton quality and fertilizability of equine oocytes

Oocyte cryopreservation is a potentially valuable technique for salvaging the germ-line when a valuable mare dies, but facilities for in vitro embryo production or oocyte transfer are not immediately available. This study examined the influence of maturation stage and freezing technique on the cryopreservability of equine oocytes. Cumulus oocyte complexes were frozen at the immature stage (GV) or after maturation in vitro for 30 hr (MII), using either conventional slow freezing (CF) or open pulled straw vitrification (OPS); cryoprotectant-exposed and untreated nonfrozen oocytes served as controls. After thawing, GV oocytes were matured in vitro, and MII oocytes were incubated for 0 or 6 hr, before staining to examine meiotic spindle quality by confocal microscopy. To assess fertilizability, CF MII oocytes were subjected to intracytoplasmic sperm injection (ICSI) and cultured in vitro. At 12, 24, and 48 hr after ICSI, injected oocytes were fixed to examine their progression through fertilization. Both maturation stage and freezing technique affected oocyte survival. The meiosis resumption rate was higher for OPS than CF for GV oocytes (28% vs. 1.2%; P < 0.05), but still much lower than for controls (66%). Cryopreserving oocytes at either stage induced meiotic spindle disruption (37%-67% normal spindles vs. 99% in controls; P < 0.05). Among frozen oocytes, however, spindle quality was best for oocytes frozen by CF at the MII stage and incubated for 6 hr post-thaw (67% normal); since this combination of cryopreservation/IVM yielded the highest proportion of oocytes reaching MII with a normal spindle (35% compared to <20% for other groups), it was used when examining the effects of cryopreservation on fertilizability. In this respect, the rate of normal fertilization for CF MII oocytes after ICSI was much lower than for controls (total oocyte activation rate, 26% vs. 56%; cleavage rate at 48 hr, 8% vs. 42%: P < 0.05). Thus, although IVM followed by CF yields a respectable percentage of normal-looking MII oocytes (35%), their ability to support fertilization is severely compromised.     

Vitrification of calf oocytes: effects of maturation stage and prematuration treatment on the nuclear and cytoskeletal components of oocytes and their subsequent development

This study was designed to establish the effects of the meiotic stage of bovine oocytes and of a prematuration treatment with roscovitine (ROS) on their resistance to cryopreservation. Oocytes from prepubertal calves at the stages of germinal vesicle breakdown (GVBD) or at metaphase II (MII) were vitrified by the open pulled straw (OPS) method. In another experiment, oocytes were kept under meiotic arrest with 50 microM ROS for 24 hr and vitrified at the GVBD stage. After warming, some oocyte samples were fixed, stained using specific fluorescent probes and examined under a confocal microscope. The remaining oocytes were fertilized, and cleavage and blastocyst rates recorded. Significantly lower cleavage rates were obtained for the vitrified GVBD and MII oocytes (9.9% and 12.6%, respectively) compared to control oocytes (73.9%). Significantly worse results in terms of cleavage rates were obtained when GVBD calf oocytes were exposed to cryoprotectants (CPAs: ethylene glycol plus dimethyl sulfoxide, DMSO) (13.1%) or vitrified (1.6%) after a prematuration treatment with ROS, when compared to untreated control oocytes (68.7%) or ROS-control oocytes (56.6%). None of the vitrification procedures yielded blastocysts, irrespective of the initial meiotic stage or previous prematuration treatment. Compared to the control oocytes, significantly fewer oocytes exhibited normal spindle configuration after being exposed to CPAs or after vitrification of either GVBD or MII calf oocytes. These results indicate that the vitrification protocol has a deleterious effect on the meiotic spindle organization of calf oocytes cryopreserved at both the GVBD and MII stage, which impairs the capacity for further development of the embryos derived from these vitrified oocytes. Prematuration treatment with ROS has no beneficial effect on the outcome of vitrification by the OPS method.     

Artificial expression of aquaporin-3 improves the survival of mouse oocytes after cryopreservation

Successful cryopreservation of mammalian cells requires rapid transport of water and cryoprotective solutes across the plasma membrane. Aquaporin-3 is known as a water/solute channel that can transport water and neutral solutes such as glycerol. In this study we examined whether artificial expression of aquaporin-3 in mouse oocytes can improve water and glycerol permeability and oocyte survival after cryopreservation. Immature mouse oocytes were injected with aquaporin-3 cRNA and were cultured for 12 h. Then the hydraulic conductivity (L(P)) and glycerol permeability (P(GLY)) of matured oocytes were determined from the relative volume changes in 10% glycerol in PB1 medium at 25 degrees C. Mean +/- SD values of L(P) and P(GLY) of cRNA-injected oocytes (3.09 +/- 1.22 micro m min(-1) atm(-1) and 3.69 +/- 1.47 x 10(-3) cm/min, respectively; numbers of oocytes = 25) were significantly higher than those of noninjected oocytes (0.83 +/- 0.02 micro m min(-1) atm(-1) and 0.07 +/- 0.02 x 10(-3) cm/min, respectively; n = 13) and water-injected oocytes (0.87 +/- 0.10 micro m min(-1) atm(-1) and 0.08 +/- 0.02 x 10(-3) cm/min, respectively; n = 20). After cryopreservation in a glycerol-based solution, 74% of cRNA-injected oocytes (n = 27) survived as assessed by their morphological appearance, whereas none of the water-injected oocytes survived (n = 10). When cRNA-injected oocytes that survived cryopreservation were inseminated in vitro, the penetration rate was 40% (n = 48) and the cleavage rate was 31% (n = 70), showing that oocytes retain their ability to be fertilized. This is the first report to show that artificial expression of a water/solute channel in a cell improves its survival after cryopreservation. This approach may enable cryopreservation of cells that have been difficult to cryopreserve.     

Morphological and biochemical analysis of immature ovine oocytes vitrified with or without cumulus cells

The cryopreservation of oocytes is an open problem as a result of their structural sensitivity to the freezing process. This study examined (i) the survival and meiotic competence of ovine oocytes vitrified at the GV stage with or without cumulus cells; (ii) the viability and functional status of cumulus cells after cryopreservation; (iii) the effect of cytochalasin B treatment before vitrification; (iv) chromatin and spindle organization; (v) the maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity of vitrified oocytes after in vitro maturation. Sheep oocytes were vitrified at different times during in vitro maturation (0, 2, and 6 h) with (COCs) or without cumulus cells (DOs). After warming and in vitro maturation, oocytes denuded at 0 h culture showed a significantly higher survival and meiotic maturation rate compared to the other groups. Hoechst 33342/propidium iodide double staining of COCs and microinjection of Lucifer Yellow revealed extensive cumulus cell membrane damage and reduced oocyte-cumulus cell communications after vitrification. Cytochalasin B treatment of COCs before vitrification exerted a negative effect on oocyte survival. After in vitro maturation, the number of vitrified oocytes with abnormal spindle and chromatin configuration was significantly higher compared to control oocytes, independently of the presence or absence of cumulus cells. The removal of cumulus cells combined with vitrification significantly decreased the MPF and MAPK levels. This study provides evidence that the removal of cumulus cells before vitrification enhances oocyte survival and meiotic competence, while impairing the activity of important proteins that could affect the developmental competence of oocytes.     

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.     

Studies on chilling sensitivity of zebrafish (Danio rerio) oocytes

Human activity in the last few decades has had a devastating effect on the diversity of fresh water and marine fish. Further decline of fish population may have serious economic and ecological consequences. One of the most promising techniques to preserve fish population is to cryopreserve their germ cells. Cryopreservation has been successfully applied to fish sperm of many species, but there has been no success with fish embryo cryopreservation and fish oocyte cryopreservation has never been studied systematically. The aim of this study is to investigate the chilling sensitivity of fish oocytes. Experiments were conducted with zebrafish stage III (vitellogenic) and stage V (mature) oocytes, which were chilled at 10, 5, 0, -5 or -10 degrees C for 15 or 60 min using a low temperature bath. Control oocytes were kept at room temperature at 22 degrees C. Oocyte viability was assessed using three different methods: trypan blue staining (TB), thiazolyl blue tetrazolium bromide (MTT) staining and observation of germinal vesicle breakdown (GVBD). The results showed that zebrafish oocyte are very sensitive to chilling and their survival decreased with decreasing temperature and increasing exposure time periods. Normalised survivals assessed with TB staining after exposure to 0, -5 or -10 degrees C for 15 or 60 min were 90.1+/-6.0, 77.8+/-7.6, and 71.2+/-9.3%, and 60.2+/-3.8, 49.6+/-6.7, and 30.4+/-3.0%, respectively. The study found that the sensitivity of viability assessment methods increase in the order of MTT < TB < GVBD. It was found that stage III oocytes were more susceptible to chilling than stage V oocytes, and that individual female had a significant influence (p < 0.0001) on oocyte chilling sensitivity. Zebrafish oocyte chilling sensitivity may also be one of the limiting factors for development of protocol of their cryopreservation.     

Membrane permeability characteristics of bovine oocytes and development of a step-wise cryoprotectant adding and diluting protocol

Membrane permeability is very helpful for the optimization of effective cryopreservation protocols. In this study, experiments were performed to determine these characteristics for immature (germinal vesicle (GV)) and in vitro matured (metaphase II (MII)) bovine oocytes within 4-37 °C, and a new step-wise adding and diluting protocol for ethylene glycol (EG) was developed and verified. Osmotically inactive volumes (V_b) of GV and MII oocytes were calculated to be 16.1% and 26.1%. The membrane permeability of the oocytes to water (L_p) in the presence of EG were between 0.08-0.18 and 0.14-0.28 μm/min/atm, and the membrane permeability of the oocytes to solutes (Ps) were between 0.0011-0.0038 and 0.0029-0.0061 cm/min for GV and MII oocytes, respectively. The activation energies (E_a) for L_p and P_s in the presence of EG were 3.68 and 6.84 kcal/mol for GV oocyte, while 3.62 and 0.83-9.08 kcal/mol for MII oocyte. The data indicated that L_p and P_s varied significantly between developmental stages and among temperatures evaluated. Based on these results, different protocols for EG adding and diluting from oocytes were developed and tested. The assessment of cleavage rate and embryonic development in vitro confirmed that the designed 4-step adding 2-step diluting protocol indicated a better outcome. The present study is helpful for better understanding of cryobiological properties and the design of cryopreservation protocols for bovine oocytes.     

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.     

First pregnancy and live birth from vitrified rabbit oocytes after intraoviductal transfer and in vivo fertilization

Intraoviductal oocyte transfer in combination with in vivo fertilization has arisen as an alternative method to induce pregnancies from cryopreserved oocytes in rabbits. In this study, offspring were obtained for the first time from vitrified rabbit oocytes using this technique. In all the experiments, recipients were artificially inseminated 9 hours before oocyte transfer. Cryopreserved (vitrified and slow-frozen) and noncryopreserved (fresh) oocytes were transferred into both oviducts, which were immediately closed using cyanoacrylate tissue adhesive to block the entry of the recipient's own oocytes. Three transferred group females that received vitrified oocytes became pregnant and delivered a total of nine live young naturally. The results revealed that there were no differences in the live birth rate between vitrified and slow-frozen oocytes (5.5% and 4.4%, respectively). When fresh oocytes were transferred, this rate increased to 19.2%, whereas in the control females (nontransferred) the rate of offspring obtained was 71.4%. This is the first reported result of the development to term of vitrified rabbit oocytes and suggests that an in vivo environment could help improve the results of oocyte cryopreservation.     

Cryopreservation of Human Oocytes and Ovarian Tissue

Oocyte cryopreservation has the potential to be an important adjunct to assisted reproductive technologies and bypasses some ethical, moral, and religious dilemmas posed by human embryo cryopreservation. The success of human oocyte cryopreservation depends on morphological and biophysical factors that could influence oocyte survival after thawing. Among the morphological factors, the maturity, quality, size of the oocyte, the presence or the absence of the cumulus oophorus seems to play an important role in oocyte survival after thawing. The main biophysical factor of cellular disruption during cryopreservation process in the intracellular ice formation that can be avoided by an adequate cell dehydration; thus reducing the intracellular water by increasing the dehydration process we can limit the damages of the cryopreservation procedure. The dehydration process can be affected by the presence and concentration of the cryoprotectants in the freezing solutions (equilibration and loading solutions), and by the freezing and thawing rate. Two additional properties of cryoprotectants help to protect cells during slow cooling, when the cells are very dehydrated and are surrounded by concentrated salts. The cryoprotectants appear to reduce damage caused by high levels of salt, a property known as salt buffering. Some events occurring to the oocyte during cryopreservation procedure has been found to be a premature exocitosis of cortical granules, leading to an intempestive zona hardening and consequently to a reduction of fertilization rate, and the cryoinjury to the zona pellucida leading to a polispermic fertilization. ICSI is an efficient method to by pass these two events and to achieve a satisfactory outcome in terms of normal fertilization of cryopreserved oocytes. The application of the ICSI to cryopreserved oocytes did not seem to increase the degeneration rate after insemination with respect to fresh oocytes. The increased oocyte survival rate and the use of ICSI have facilitated the recent increase in the number of pregnancies and live birth.