Cryopreservation of mouse spermatozoa

Recently, it has become possible to freeze a large number of mouse spermatozoa immediately after collection from the epididymides of a small number of males. The cryopreservation of spermatozoa is simpler, less time-consuming, and less costly than that of embryos for maintaining various strains of mice with induced mutations. This chapter attempts to provide a realistic overview of the cryopreservation of mouse spermatozoa and to describe a detailed procedure for mouse sperm freezing. Received: 16 December 1999 / Accepted: 17 December 1999     

Survival of rapidly frozen hatched mouse blastocysts

The objective of the present study was to examine the effect of rapid freezing on the in vitro and in vivo survival of zona-pellucida-free hatched mouse blastocysts. Hatched blastocysts were rapidly frozen in a freezing medium containing either ethylene glycol (EG) or glycerol (G) in 1.5 M or 3 M concentration. Prior to freezing, embryos were equilibrated in the freezing medium for 2 min, 10 min, 20 min or 30 min at room temperature. To freeze them, embryos were held in liquid nitrogen vapour [approximately 1 cm above the surface of the liquid nitrogen (LN2)] for 2 minutes and then immersed into LN2. After thawing, embryos were transferred either to rehydration medium (DPBS + 10% foetal calf serum +0.5 M sucrose) for 10 minutes or rehydrated directly in DPBS supplemented with foetal calf serum. In vitro survival of embryos frozen with EG was higher than those frozen with G. The highest survival was obtained with 3 M EG and 2 min or 10 min equilibration prior to freezing, combined with direct rehydration after thawing. Frozen blastocysts developed into normal foetuses as well as unfrozen control ones did, with averages of 30% (control), 26% (EG) and 15% (G). The results show that hatching and hatched mouse blastocysts can be cryopreserved by a simple rapid freezing protocol in EG without significant loss of viability. Our data indicate that the mechanical protection of the zona pellucida is not needed during freezing in these stages.     

经程序化冷冻的小鼠休眠胚胎的基因表达谱差异分析

目的探讨小鼠休眠胚胎经程序化冷冻后基因表达谱的变化及相关信号通路的改变趋势。方法采用Affymetrix基因芯片检测小鼠正常休眠胚胎和经程序化冷冻后的休眠胚胎的差异表达基因;采用GO分析和Pathway分析等生物信息学方法进一步了解相关信号通路的改变。结果经程序化冷冻后的小鼠休眠胚胎与正常休眠胚胎相比,存在228个差异表达基因,其中50个基因表达上调,178个基因表达下调。Pathway分析显示黏着斑通路、细胞外基质受体相互作用通路、肌动蛋白细胞骨架调节通路、细胞凋亡通路、细胞通讯通路、泛素介导的蛋白质水解通路、甘油磷脂代谢通路、小细胞肺癌通路、TGF-β信号通路、MAPK信号通路等基因差异表达变化趋势明显。结论小鼠休眠胚胎经程序化冷冻后会导致一系列基因调控变化,并可能影响多条信号通路的协同变化。     

Media alternatives for the collection, culture and freezing of mouse and cattle embryos

The replacement of biological products in media for the collection, culture and freezing of mammalian embryos was studied. To test the hypothesis that chemically defined surfactants can replace bovine serum albumin (BSA) or serum in embryo media, morula-stage mouse and cattle embryos were collected, cultured, and/or frozen in the surfactant compound, VF5. Collection efficiency of mouse and cattle embryos did not differ whether the medium contained serum or surfactant. In addition, morula-stage mouse and cattle embryos developed and hatched at similar rates in culture media containing either BSA or surfactant. Although the freeze/thaw survival and development in culture of bovine embryos was not significantly different in any of the media, there was a significantly lower hatching rate of mouse embryos frozen with serum or surfactant than with cryoprotectant alone or with cryoprotectant plus albumin-free serum. However, the absence of serum or surfactant in embryo freezing media resulted in embryo loss, presumably due to stickiness. The data suggest that serum can be replaced by a chemically defined surfactant in mouse and cattle embryo transfer systems for the collection, culturing and freezing of embryos. It is likely that the beneficial effects of serum are due to its surfactant properties.     

Effect of osmolality and oxygen tension on the survival of mouse sperm frozen to various temperatures in various concentrations of glycerol and raffinose

Cryopreserved mouse sperm are beginning to be used to meet the demand of a reliable cost-effective method for maintaining the rapidly expanding numbers of lines of mutant mice. However, successful and reproducible cryopreservation has proven to be a difficult problem. Furthermore, the underlying factors responsible for success or failure are mostly obscure. Several contributors to these difficulties have been identified. Our laboratory has found that mouse sperm are extremely susceptible to the mechanical stresses associated with pipetting, mixing, and centrifugation, and others have found that they are severely limited in their tolerance to osmotic volume changes. We have hypothesized two other contributors to the difficulties. One is that the concentrations of glycerol used in published protocols are substantially lower than those found to be optimal for most mammalian cells. The other hypothesis relates to the fact that mouse sperm membranes are especially susceptible to damage from oxygen-derived free radicals. That damage may reduce their ability to survive freezing. If so, survival ought to increase if the concentration of oxygen is kept low throughout the procedure. To achieve low levels, we have incorporated an Escherichia coli membrane fraction, Oxyrase, into all media. A previous report showed a protective effect. That is confirmed here under a broader range of conditions. The conditions studied have been the individual and interactive effects of the concentrations of glycerol, raffinose, and phosphate-buffered saline (PBS) on motility after freezing at 21 degrees C/min to -70 degrees C. Cryoprotection increased with increasing raffinose concentration, provided that the concentration of PBS was appropriately reduced to hold the total osmolality of nonpermeating solutes to within tolerated limits. Surprisingly, the best results were achieved in the total absence of glycerol. The highest motilities to date (68 +/- 8%) after freezing to -70 degrees C have been achieved using media containing Oxyrase, 0 M glycerol, and 18% raffinose in 14x strength modified PBS. We also determined the motility loss after freezing to intermediate temperatures, i.e., -10 and -30 degrees C. The major motility loss occurred by -10 degrees C, especially in the absence of Oxyrase. These results suggest that a major problem in the freezing of mouse sperm is the physical stress resulting from extracellular ice crystal formation. Oxyrase appears to lessen that damage substantially.     

Quick freezing of one-cell mouse embryos using ethylene glycol with sucrose

One-cell mouse embryos were frozen by direct plunging into liquid nitrogen (LN(2)) vapor after equilibration in 3 M ethylene glycol with 0.25 M sucrose (freezing medium) for 5 to 40 minutes. After thawing, the embryos were cultured in vitro and the effects of the equilibration period and dilution method were examined. No significant difference was observed in the in vitro survival of embryos when 0.5 or 1.0 M sucrose was used for the dilution of the cryoprotectant for each equilibration period. The highest survival rate (67.2%) was obtained when the embryos were equilibrated for 10 minutes, and the cryoprotectant diluted with either 0.5 or 1.0 M sucrose after thawing. Shorter (5 minutes) or prolonged (40 minutes) equilibration of embryos in the freezing medium yielded significantly lower survival rates. Dilution by direct transfer of the frozen-thawed embryos into PB1 resulted in lower survival rates than when 0.5 or 1.0 M sucrose was used. The in vitro development to the blastocyst stage of one-cell mouse embryos frozen after 10 minutes equilibration in the freezing medium and diluted after thawing in 0.5 M sucrose was significantly lower than the control (68.0 vs 92.7%). However, transfer of the blastocysts developing from frozen-thawed one-cell mouse embryos into the uterine horns of the recipients resulted in fetal development and implantation rates similar to the control.     

冻融后小鼠休眠胚胎超微结构的变化

目的从亚细胞超微结构的角度揭示其抗冻能力优于正常孵化胚胎的原因。方法利用透射电子显微镜观察小鼠休眠胚胎与正常孵化期胚胎在细胞连接和各细胞器形态与分布上的差异,以及冻融培养后的变化,并进行相关比较分析。结果通过亚细胞结构对比分析发现:冷冻前小鼠休眠胚胎为紧缩状,处于能量代谢较低的"基态",通过冻融后培养,细胞器结构恢复与正常孵化胚胎冷冻前相似;而正常孵化胚胎经过冻融后,线粒体数量减少,细胞核松散,异染色质增多。结论小鼠休眠胚胎与正常孵化胚胎冻融后相比,其细胞状态更有利于物质储存及能量代谢,表明小鼠休眠胚胎从亚细胞超微结构的角度比其正常孵化胚胎更具抗冻性。     

Microscopic observation of intracellular ice formation in unfertilized mouse ova as a function of cooling rate

A physical-chemical analysis of water loss from cells at subzero temperatures had shown that the likelihood of intracellular ice formation increased with increasing cooling rate (22). We have now used a modified version of a unique conductioncooled cryomicroscope stage (8) to observe the freezing of unfertilized mouse ova suspended in dimethyl sulfoxide. Survival measurements showed that the respective survivals of ova were about 65, 56, and 0% when they were cooled at rates of 0.2 to 1.5, 2.5, and 5.4 °C/min. Direct microscopic observation of mouse ova during freezing showed that the respective fractions of cells that froze intracellularly were 13, 72, and 100% when they were cooled at rates of 1.3, 2.9, and 4.8 °C/min or faster. These values agree with those predicted from the physical-chemical analysis for cells the size of mouse ova. The microscopic observations have also shown that intracellular freezing generally occurred at about ?40 to ?45 °C. We had previously observed that mouse embryos must be cooled slowly to ?50 °C or below if they are to survive subsequent rapid cooling to ?196 °C. The observation of intracellular ice formation at ?45 °C supports the interpretation that at temperatures above ?50 °C the embryos still contain water capable of freezing intracellularly.     

Comparison of DNA apoptosis in mouse and human blastocysts after vitrification and slow freezing

Vitrification is a novel cryopreservation method for mammalian blastocysts. This study was designed to compare different vitrification methods and slow freezing for their effects on survival rate and DNA integrity in mouse and human blastocysts. In Experiment 1, embryo survival and DNA integrity were compared between mouse blastocysts with collapsed and non‐collapsed blastoceles. In Experiment 2, embryo survival and DNA integrity were compared between vitrified and slow‐frozen mouse blastocysts. In Experiment 3, embryo survival and DNA integrity were compared between vitrified and slow‐frozen human blastocysts. Fresh blastocysts were used as controls in all experiments. Higher (P < 0.05) blastocyst survival rates were obtained in mouse blastocysts vitrified with collapsed versus intact blastoceles, although DNA‐integrity indices in the surviving blastocysts were the same among vitrified and fresh blastocysts. More mouse blastocysts (P < 0.05) survived after vitrification (100%) as compared to slow freezing (82.5%). DNA‐integrity indices examined in the surviving blastocysts were also higher (P < 0.001) in fresh (93.6%) and vitrified/warmed (93.7%) blastocysts than in slow‐frozen/thawed (75.8%) ones. More human blastocysts survived with a higher DNA‐integrity index after vitrification/warming than after slow freezing/thawing. These results indicate that higher survival rates can be obtained by vitrification of blastocele‐collapsed blastocysts, and that vitrification causes less cell apoptosis in both mouse and human blastocysts compared to slow freezing. Vitrification of blastocysts after blastocele collapse by single laser pulse supports a higher survival rate and less DNA apoptosis, suggesting that laser blastocele collapse is a safe procedure for blastocyst vitrification. Mol. Reprod. Dev. 79: 229–236, 2012. © 2011 Wiley Periodicals, Inc.     

Oocyte activation after intracytoplasmic injection with sperm frozen without cryoprotectants results in live offspring from inbred and hybrid mouse strains

Re-establishment of mouse strains used for mutagenesis and transgenesis has been hindered by difficulties in freezing sperm. The use of intracytoplasmic sperm injection (ICSI) enables the production of embryos for the restoration of mouse lines using sperm with reduced quality. By using ICSI, simplified sperm-freezing methods such as snap freezing can be explored. We examined the capacity of embryos from the inbred C57Bl/6J and 129Sv/ImJ mouse strains, commonly used for transgenic and N-ethyl-N-nitrosourea mutagenesis purposes to develop to blastocysts in vitro and to term following ICSI with sperm frozen without cryoprotectant. The results were compared to F1 (C57BlxCBA) hybrid embryos. Following freezing, sperm were immotile but could fertilize oocytes at similar rates to fresh sperm. However, embryo development in vitro to the blastocyst stage was reduced in all three strains. No pups were born from C57Bl/6J or 129Sv/ImJ embryos obtained from frozen sperm following transfer to foster females, and only a limited number of F1 embryos developed to term. Activation of oocytes injected with frozen sperm with 1.7 mM Sr2+ (SrCl2) did result in the birth of pups in all three strains. We conclude that the inability of sperm frozen without cryoprotectants to effectively activate oocytes may affect embryo development to term and can be overcome by strontium activation. This may become an effective strategy for sperm preservation and the restoration of most popular strains used for genetic modifications.     

Cultivation of frozen mouse bone marrow cells in agar.

The authors attempted to cultivate frozen mouse bone marrow cells in a semisolid medium. They demonstrated that the stem haematopoietic cells of frozen mouse bone marrow were capable of proliferation and of colony formation on agar. The much smaller number of colonies from frozen mouse bone marrow (about 80% fewer) compared with fresh marrow is evidence that part of the stem haematopoietic cell population retains proliferative capacity even after freezing.     

Effects of hold time after extracellular ice formation on intracellular freezing of mouse oocytes

MII mouse oocytes in 1 and 1.5M ethylene glycol(EG)/phosphate buffered saline have been subjected to rapid freezing at 50 degrees C/min to -70 degrees C. When this rapid freezing is preceded by a variable hold time of 0-3 min after the initial extracellular ice formation (EIF), the duration of the hold time has a substantial effect on the temperature at which the oocytes subsequently undergo intracellular ice formation (IIF). For example, in 1M EG, the IIF temperatures are -23.7 and -39.2 degrees C with 0 and 2 min hold times; in 1.5M EG, the corresponding IIF temperatures are -29.1 and -40.8 degrees C.     

Large numbers of mice established by in vitro fertilization with cryopreserved spermatozoa: implications and applications for genetic resource banks, mutagenesis screens, and mouse backcrosses

Recent advances in the methodologies for cryopreservation of mouse spermatozoa have opened up a number of opportunities for mouse geneticists. We have investigated various applications for this relatively new technology and have explored the potential of sperm freezing coupled with IVF for archiving, stock building, and the rapid establishment of backcrosses. Firstly, we investigated the use of sperm freezing for the archiving of (C3H/HeH × BALB/c)F₁ progeny from a large-scale mutagenesis program. We have demonstrated that it is possible to establish efficient, comprehensive, and deep archives and that potentially thousands of offspring can be derived from the frozen spermatozoa of a single mutant male mouse. Secondly, we examined the efficacy of sperm freezing for a number of other genotypes. For at least some genotypes, frozen spermatozoa can be utilized to rapidly build stock far more quickly than by conventional methods. Finally, we demonstrated that it is feasible to use frozen spermatozoa from the mouse mutant archive for the rapid generation of mutant backcross progeny. Received: 8 February 1999 / Accepted: 5 May 1999     

Effectiveness of slow freezing and vitrification for long-term preservation of mouse ovarian tissue

This study was conducted to evaluate the interaction between cryo-damage and ART outcome after cryopreservation of mouse ovarian tissues with different methods. Either a vitrification or a slow freezing was employed for the cryopreservation of B6CBAF1 mouse ovaries and follicle growth and the preimplantation development of intrafollicular oocytes following parthenogenesis or IVF were monitored. Both cryopreservation protocols caused significant damage to follicle components, including vacuole formation and mitochondrial deformities. Regardless of the cryopreservation protocols employed, a sharp (P < 0.0001) decrease in follicle viability and post-thaw growth was detected. When IVF program was employed, significant (P < 0.05) decrease in cleavage and blastocyst formation was notable in both modes of cryopreservation. However, such retardation was not found when oocytes were parthenogenetically activated. In the IVF oocytes, slow freezing led to better development than vitrification. In conclusion, a close relationship between cryopreservation and ART methods should be considered for the selection of cryopreservation program.     

Nonequilibrium freezing of one-cell mouse embryos. Membrane integrity and developmental potential.

A thermodynamic model was used to evaluate and optimize a rapid three-step nonequilibrium freezing protocol for one-cell mouse embryos in the absence of cryoprotectants (CPAs) that avoided lethal intracellular ice formation (IIF). Biophysical parameters of one-cell mouse embryos were determined at subzero temperatures using cryomicroscopic investigations (i.e., the water permeability of the plasma membrane, its temperature dependence, and the parameters for heterogeneous IIF). The parameters were then incorporated into the thermodynamic model, which predicted the likelihood of IIF. Model predictions showed that IIF could be prevented at a cooling rate of 120 degrees C/min when a 5-min holding period was inserted at -10 degrees C to assure cellular dehydration. This predicted freezing protocol, which avoided IIF in the absence of CPAs, was two orders of magnitude faster than conventional embryo cryopreservation cooling rates of between 0.5 and 1 degree C/min. At slow cooling rates, embryos predominantly follow the equilibrium phase diagram and do not undergo IIF, but mechanisms other than IIF (e.g., high electrolyte concentrations, mechanical effects, and others) cause cellular damage. We tested the predictions of our thermodynamic model using a programmable freezer and confirmed the theoretical predictions. The membrane integrity of one-cell mouse embryos, as assessed by fluorescein diacetate retention, was approximately 80% after freezing down to -45 degrees C by the rapid nonequilibrium protocol derived from our model. The fact that embryos could be rapidly frozen in the absence of CPAs without damage to the plasma membrane as assessed by fluorescein diacetate retention is a new and exciting finding. Further refinements of this protocol is necessary to retain the developmental competence of the embryos.     

Piezo-actuated mouse intracytoplasmic sperm injection (ICSI)

The mouse is a genetically tractable model organism widely used to study mammalian development and disease. However, mouse metaphase II (mII) oocytes are exquisitely sensitive and intracytoplasmic sperm injection (ICSI) with conventional pipettes generally kills them. This problem can be solved with piezo-actuated micromanipulation, in which the piezo-electric effect (crystal deformation in response to an externally applied voltage) propels a microinjection needle tip forward in a precise and rapid movement. Piezo-actuated micromanipulation enhances the penetration of membranes and matrices, and mouse ICSI is a major application. Here we describe a comprehensive, step-by-step mouse piezo ICSI protocol for non-specialists that can be completed in 2-4 h. The protocol is a basic prelude to multiple applications, including nuclear transfer cloning, spermatid injection, blastocyst injection, mII transgenesis, and streamlining micromanipulation in primates and livestock. Moreover, piezo ICSI can be used to obtain offspring from 'dead' (non-motile) sperm, enabling trivial sperm freezing protocols for mouse strain storage and shipment.     

Short-term preservation of mouse oocytes at 5 degrees C.

The temporary preservation of oocytes without freezing would be useful for some experiments. ICR mouse oocytes were kept in a preservation medium under mineral oil for 1, 2, 3, 4 or 7 days at 5 degrees C, and 1 or 2 days at 37 degrees C. In vitro fertilization was attempted on oocytes rinsed with TYH medium after preservation. More than 70% of morphologically normal oocytes were recovered from each preservation group. Fertilization rates of oocytes preserved for 1, 2, 3, 4 or 7 days at 5 degrees C were 69.9, 66.5, 45.3, 26.7 and 8.8% respectively. Fertilization rates of oocytes preserved for 1 or 2 days at 37 degrees C were 9.6 and 1.6%, respectively. Preservation of oocytes at 5 degrees C has some capability as a method of short-term storage without freezing.     

Preservation of mouse sperm by convective drying and storing in 3-O-methyl-D-glucose

With the fast advancement in the genetics and bio-medical fields, the vast number of valuable transgenic and rare genetic mouse models need to be preserved. Preservation of mouse sperm by convective drying and subsequent storing at above freezing temperatures could dramatically reduce the cost and facilitate shipping. Mouse sperm were convectively dried under nitrogen gas in the Na-EGTA solution containing 100 mmol/L 3-O-methyl-D-glucose and stored in LiCl sorption jars (Relative Humidity, RH, 12%) at 4°C and 22°C for up to one year. The functionality of these sperm samples after storage was tested by intracytoplasmic injection into mouse oocytes. The percentages of blastocysts produced from sperm stored at 4°C for 1, 2, 3, 6, and 12 months were 62.6%, 53.4%, 39.6%, 33.3%, and 30.4%, respectively, while those stored at 22°C for 1, 2, and 3 months were 28.8%, 26.6%, and 12.2%, respectively. Transfer of 38 two- to four-cell embryos from sperm stored at 4°C for 1 year produced two live pups while 59 two- to four-cell embryos from sperm stored at 22°C for 3 months also produced two live pups. Although all the pups looked healthy at 3 weeks of age, normality of offspring produced using convectively dried sperm needs further investigation. The percentages of blastocyst from sperm stored in the higher relative humidity conditions of NaBr and MgCl(2) jars and driest condition of P(2)O(5) jars at 4°C and 22°C were all lower. A simple method of mouse sperm preservation is demonstrated. Three-O-methyl-D-glucose, a metabolically inactive derivative of glucose, offers significant protection for dried mouse sperm at above freezing temperatures without the need for poration of cell membrane.     

Cultivation of fresh and frozen mouse bone marrow. - Application of methyl cellulose.

The culture of cells of both fresh and frozen mouse bone marrow on methyl cellulose (MC) was approached. We used 1.5%-concentration of MC and proved the stem cells of fresh and frozen mouse bone marrow to proliferate and form haemopoietic colonies on MC. We established that the freezing process did not significantly decrease the proliferative capacity of CFU-C stem cells.     

Frozen fetal and neonatal mouse cardiac reimplants for assessing cryoprotective agents

The electrical activity of transplanted syngeneic 17–19 day fetal and 1–9 day neonatal mouse hearts has been studied after freezing to −196 °C in the presence of different cryoprotective agents. Histological examinations were performed after electrical activity (QRS) had been studied for periods in excess of 30 days. EG, DMSO, and glycerol appear equally protective provided that glycerol is added at 37 °C. Methanol, although nontoxic at those concentrations which will protect tissue culture cells, does not offer protection to this type of organized tissue. None of the high molecular weight compounds tested offered cryoprotection in this system. Diffusion of both protective agents and nutrients has been shown to be limiting factors in the survival of frozen-thawed neonatal hearts between 6 and 9 days of age.