Cryopreservation of Human Embryos

Cryopreservation of human embryos from the 2-cell stage up to the morula stage is a safe procedure which has been carried out for the last 25 years. Experience with blastocyst cryopreservation is still limited and pregnancy rates after the use of frozen, thawed blastocysts vary extremely. Vitrification has improved the success of embryo cryopreservation. However, this technique cannot yet be considered as a routine procedure. Despite all of the advantages for infertile couples, cryopreservation of human embryos creates severe ethical problems, because of surplus frozen embryos which either have to be destroyed or perhaps used for research. Embryo adoption may provide a solution to solve imminent medical, ethical and social problems.     

鲈鱼胚胎的玻璃化冷冻保存

本文对鲈鱼(Lateolabrax japonicus)胚胎进行了玻璃化冷冻保存研究,筛选出了浓度较低、玻璃化程度较稳定的5种玻璃化液,冷冻时形成玻璃化的概率在48．1％～100％,在35～43℃的水浴中解冻时保持玻璃化的概率在44．4％～63．0％;玻璃化液VSD2在解冻时保持玻璃化的概率最高。对鲈鱼神经胚、20对肌节胚、尾芽胚、心跳胚、出膜前胚在玻璃化液VSD2中的适应能力及适合于玻璃化冷冻的胚胎时期进行了比较,结果显示：不同时期胚胎对玻璃化液的耐受能力不同,鲈鱼神经胚耐受能力最低,心跳胚耐受能力最强,出膜前期胚次之,心跳胚和出膜前胚适合于进行玻璃化冷冻。对0．5mol/L蔗糖的洗脱时间进行了选择,结果显示,洗脱10～20min效果较好。利用玻璃化程度较好的VSD2对鲈鱼不同时期胚胎进行超低温(-196℃)冷冻,获得了2．1％～27．9％的透明胚。将鲈鱼心跳胚冷冻解冻后获2粒复活胚,培养至出膜期,成活42～50h;出膜前期胚在冷冻解冻后有1粒胚复活,并且孵化出鱼苗[动物学报49(6)：843～850,2003]。     

A chronologic review of mature oocyte vitrification research in cattle, pigs, and sheep

Vitrification as a means of cryopreservation has become a standard approach for oocytes from livestock. This paradigm shift occurred primarily as a result of the demonstration in 1996 that bovine oocytes are extremely susceptible to chilling injury. Since that early work, numerous devices have been used as supports for oocytes during so-called “ultra-rapid cooling”, and occasionally, trials involving the deposition of small volumes of media containing oocytes directly into liquid nitrogen to facilitate cooling have been reported. Results reporting blastocyst development exceeding 10% are common, but variability remains high, and a standard method for bovine oocytes remains to be established. Oocytes from pigs are particularly difficult to cryopreserve, even with the use of ultrarapid cooling approaches. Few reports have demonstrated blastocyst development exceeding 5%. The application of hydrostatic pressure before vitrification appears to impart stress tolerance to porcine oocytes, as the results of some treatments have shown development to blastocysts at proportions >10%. Work on sheep oocyte vitrification is relatively new, and a few articles have reported blastocyst development at 10% or more. Messenger RNA levels are reportedly altered in sheep oocytes as a result of vitrification, and damage to the cytoskeleton is common across species.     

一种安全有效的卵母细胞封闭式玻璃化冷冻载体

目的探讨封闭式玻璃化冷冻载体冻存小鼠卵母细胞的可行性。方法以小鼠MII期卵母细胞为模型,以开放式玻璃微细管法（GMP）为对照组,比较两种玻璃化冷冻载体对小鼠卵母细胞冷冻后的存活率、受精率、卵裂率及囊胚率的影响。结果卵母细胞经冻融后,封闭式冷冻载体组和GMP组的存活率、受精率、卵裂率和囊胚率均没有明显差异（92.80%vs93.11%,49.80%vs51.67%,36.73%vs35.83%,12.65%vs14.17%%;P〉0.05）。结论封闭式冷冻载体能安全、有效的冷冻保存小鼠卵母细胞。     

衣藻细胞玻璃化超低温保存技术的研究

本研究以衣藻为材料,探讨其玻璃化超低温保存的条件和方法,结果表明,衣藻经含0.25mol/L蔗糖溶液的TAP培养基预培养一天后,在玻璃化冷冻保护剂中脱水5分钟,直接投稿液氮,48小时后快速化冻,去保护剂并用含0.5mol/L蔗糖溶液的TAP培养基境培养一天,再转到ATP培养基暗培养一天,最后置光照条件下恢复培养,其存活率可达31.45％,恢复培养后衣藻细胞的生长规律与未冻存的衣藻相一致。     

Vitrification of turbot embryos: preliminary assays

Successful fish embryo cryopreservation is still far from being achieved. Vitrification is considered the most promising option. Many factors are involved in the success of the process. The choice of a proper vitrification solution, the enzymatic permeabilization of embryos to increase cryoprotectant permeability, the adequate container for embryo loading, and the temperature for thawing, were the parameters considered at different developmental stages in the present study. After vitrification, embryo morphology was evaluated under stereoscopic microscopy, establishing the percentage of intact embryos. Two of the studied parameters yielded differences in this percentage, the volume of straw used for embryo loading (1 ml straws were significantly better than 0.5 ml straws, with regard to post-thawed embryo morphologies), and the thawing temperature, achieving 49% of embryos with intact morphology after thawing at 0 degrees C. After thawing, the intact embryos were incubated and periodically observed to detect morphological changes. Changes in the perivitelline space, shrinkage of the yolk and chorion ruptures as well as a progressive whitening of the embryo and yolk were observed. After 8 h all embryos showed clear signs of degradation and during this incubation period no embryo showed any developmental ability.     

拟南芥悬浮细胞系的玻璃化法超低温保存

悬浮培养细胞系是植物生理生化研究的好材料之一。为了保持细胞系的遗传稳定性,需要采用超低温保存技术。玻璃化法是一种不用程序降温仪的超低温保存技术。本文报道了从模式植物拟南芥建立悬浮细胞系并对其进行玻璃化法超低温研究。细胞经过合理的预培养处理和保护剂处理,直接投入液氮贮存。复温后的细胞能恢复生长,恢复生长的细胞保持着植株再生能力。国外,拟南芥悬浮细胞系的程序降温法保存和包埋脱水法保存已经报道,玻璃化法保存尚未见报道。     

Identification of a novel Xenopus laevis poly (A) binding protein

Poly (A) binding proteins are intimately implicated in controlling a number of events in mRNA metabolism from nuclear polyadenylation to cytoplasmic translation and stability. The known poly(A) binding proteins can be divided into three distinct structural groups (prototypes PABP1, PABPN1/PABP2 and Nab2p) and two functional families, showing that similar functions can be accomplished by differing structural units. This has prompted us to perform a screen for novel poly(A) binding proteins using Xenopus laevis. A novel poly(A) binding protein of 32 kDa (p32) was identified. Sequence analysis showed that p32 has about 50% identity to the known nuclear poly(A) binding proteins (PABPN1) but is more closely related to a group of mammalian proteins of unknown function. The expression of Xenopus laevis ePABP2 is restricted to early embryos. Accordingly, we propose that p32 is the founder member of a novel class of poly(A) binding proteins named ePABP2.     

小鼠卵慢速和快速降温及玻璃化冻存的比较研究

本文用４种冷冻模式：慢速降温（ＳＬ）,超快速降温（Ｑ１）和防冻剂组成不同的玻璃化法（Ｒ－ＦＶＭ和ＭＶＭ）对昆明小鼠卵进行冷冻保存的比较研究,并用体外受精技术检测冷冻复苏卵的受精能力。ＳＬ和Ｒ－ＦＶＭ的存活率分别为５５．１±１．２和６５．９±７．９,显著高于Ｑ１（２４．２±７．３）和ＭＶＭ（４．５±２．４）其受精率分别为７２．８±１．８和７３．９±０．４显著高于Ｑ１（５８．６±１１．２）和ＭＶＭ（４１．５±８．５）,而与对照组（７７．５±３．９）相似。结果表明：１）慢速降温程序（ＳＬ）和同时含有渗透性和非渗透性防冻剂的玻璃化法（Ｆ—ＲＶＭ）较适宜昆明小鼠卵的冻存;２）同时考虑冻存过程中的冰晶损伤和渗透压损伤是获得较好冻存结果的关键。     

Vitrification of early-stage bovine and equine embryos

The objectives of this study were to: (1) determine an optimal method and stage of development for vitrification of bovine zygotes or early embryos; and (2) use the optimal procedure for bovine embryos to establish equine pregnancies after vitrification and warming of early embryos. Initially, bovine embryos produced by in-vitro fertilization (IVF) were frozen and vitrified in 0.25 mL straws with minimal success. A subsequent experiment was done using two vitrification methods and super open pulled straws (OPS) with 1- or 8-cell bovine embryos. In Method 1 (EG-O), embryos were exposed to 1.5 M ethylene glycol (EG) for 5 min, 7 M ethylene glycol and 0.6 M galactose for 30 s, loaded in an OPS, and plunged into liquid nitrogen. In Method 2 (EG-DMSO), embryos were exposed to 1.1 M ethylene glycol and 1.1 M dimethyl sulfoxide (DMSO) for 3 min, 2.5 M ethylene glycol, 2.5 M DMSO and 0.5 M galactose for 30 s, and loaded and plunged as for EG-O. Cryoprotectants were removed after warming in three steps. One- and eight-cell bovine embryos were cultured for 7 and 4.5 d, respectively, after warming, and control embryos were cultured without vitrification. Cleavage rates of 1-cell embryos were similar (P > 0.05) for vitrified and control embryos, although the blastocyst rates for EG-O and control embryos were similar and higher (P < 0.05) than for EG-DMSO. The blastocyst rate of 8-cell embryos was higher (P < 0.05) for EG-O than EG-DMSO. Therefore, EG-O was used to cryopreserve equine embryos. Equine oocytes were obtained from preovulatory follicles. After ICSI, injected oocytes were cultured for 1-3 d. Two- to eight-cell embryos were vitrified, warmed and transferred into recipient's oviducts. The pregnancy rate on Day 20 was 62% (5/8) for equine embryos after vitrification and warming. In summary, a successful method was established for vitrification of early-stage bovine embryos, and this method was used to establish equine pregnancies after vitrification and warming of 2- to 8-cell embryos produced by ICSI.     

Calcium concentration in vitrification medium affects the developmental competence of in vitro matured ovine oocytes

The present study was designed to determine whether different calcium concentrations in the vitrification solutions could improve the developmental competence of in vitro matured ovine oocytes after cryopreservation. In vitro matured oocytes were vitrified with 16.5% ethylene glycol (EG) + 16.5% dimethylsulfoxide (DMSO) vitrification media. The base media contain different calcium concentrations, so that five experimental groups were obtained: TCM/FCS (TCM 199 + 20% fetal calf serum (FCS), [Ca²⁺] 9.9 mg/dl); PBS/FCS (Dulbecco Phosphate Buffered Saline (PBS) + 20% FCS, [Ca²⁺] 4.4 mg/dl); PBS^{CaMg free}/FCS (PBS without Ca²⁺ and Mg²⁺ + 20% FCS [Ca²⁺] 2.2 mg/dl); PBS/BSA (PBS + 0.4% bovine serum albumin (BSA), [Ca²⁺] 3.2 mg/dl) and PBS^{CaMg free}/BSA (PBS without Ca²⁺ and Mg²⁺ +0.4% BSA, [Ca²⁺] 0.4 mg/dl). After warming, the oocytes from the five experimental groups were assessed for survival, spontaneous parthenogenetic activation and developmental capacity via in vitro fertilization. Oocyte survival after vitrification procedures was better preserved in group PBS^{CaMg free}/FCS compared to the others (P < 0.05). In addition, a positive correlation was found between calcium concentration in vitrification solutions and spontaneous parthenogenetic activation (correlation index 0,82; P < 0.001). Development of vitrified oocytes was significantly affected by vitrification media composition (P < 0.01). In particular, oocytes from group PBS^{CaMg free}/FCS led to higher cleavage rates and blastocyst rate compared to the others. Our data showed that lowering calcium concentration in the vitrification medium improves the blastocyst rate of vitrified ovine oocytes, probably reducing the effect of EG + DMSO during vitrification. On the contrary, the replacement of FCS with BSA dramatically reduces the developmental potential of these oocytes.     

香蕉茎尖超低温保存过程中的细胞超微结构观察(简报)

超低温保存(Cryopreservation)通常称为液氮保存或LN(-196℃)保存,是目前植物种质资源长期稳定保存的理想方法,已经成功应用于多种植物种质资源保存。玻璃化法(Vitrification)超低温保存植物种质资源始于20世纪80年代末,Uagami等首次