哺乳动物的卵巢冷冻保存

经冷冻保存后的卵巢或组织可以保存大量的原始卵泡,并且结构完整,具有活力及发育能力,且已直接用于临床获得妊娠。因此,卵巢冷冻近年来成了生殖生物学、生殖医学的研究热点。本文就卵巢冷冻的历史、基本技术原理、方案选择与评价及其影响因素作一综述。     

哺乳动物卵母细胞的超低温冷冻保存

叙述了哺乳动物卵母细胞超低温冷冻保存的研究进展，分析了影响卵母细胞超低温冷冻保存后存活率的关键因素，推荐了较为成效的卵母细胞超低温冷冻保存方法，认为哺乳动物卵母细胞低温保存技术的应用潜力巨大。     

哺乳动物胚胎超低温冷冻保存的研究进展

哺乳动物胚胎超低温保存最早在小鼠获得成功。继之 ,牛、兔、绵羊、大鼠、山羊、猪、人等的胚胎冷冻保存成功并取得了很大进展[1～ 4 ] 。194 9年Ｐｏｌｇｅ冷冻哺乳动物精子获得成功 ,发现了甘油在精子冷冻过程中能起到保护作用[5] 。 1972年 ,英国学者Ｗｈｉｔｔｉｎｇｈａｍ首先成功地冷冻保存了小鼠胚胎 ,发明了胚胎的慢速冷冻保存方法 ,这项技术一直被广泛应用[6] 。 1973年Ｗｉｌｍｕｔ和Ｒｏｕｓｏ以甘油为防冻剂保存牛胚胎成功[5] 。 1985年Ｒａｌｌ和Ｆａｈｙ又发明了胚胎的玻璃化冷冻保存技术 ,该技术的开发 ,简化了操作程序 ,大…     

卵巢冷冻保存和移植的历史、现状及前景

随着卵巢组织冷冻保存方法和冷冻技术的发展,生殖系的保存已变成了现实,卵巢组织冷冻和移植将作为一种保存种质资源最长远的选择,如冻存卵巢组织移植为年轻女性患者恢复内分泌及生育能力提供了可能.试验表明,冻存卵巢移植后其功能可恢复,并能保持较长期功能.文章就卵巢冷冻保存和卵巢移植技术的发展进步,卵巢组织冷冻和移植的历史、研究进展、方法、影响因素及临床应用进行综述.     

哺乳动物胚胎玻璃化冷冻保存的研究进展

玻璃化(vitrification)是指利用物理学原理将高浓度的抗冻保护剂急速降温后，由液态转化为外形类似玻璃状的稳定而透明的非晶体化固体状态的过程。玻璃化的固体保留了液体正常的分子和离子分布，可以视为一种无序黏稠的超冷液体，因而避免了细胞内形成冰晶而对细胞产生伤害。1985年，Rall等率先将玻璃化冷冻方法用于小鼠胚胎的冷冻保存，推动了该方法在哺乳动物胚胎冷冻保存的方面的应用。之后，玻璃化冷冻保存不断改进，     

哺乳动物生殖细胞冷冻保存对其DNA甲基化影响的研究进展

哺乳动物生殖细胞和胚胎冷冻保存在辅助生殖技术中得到了广泛的应用,同时在保存物种的多样性方面发挥着巨大的作用。但是,冷冻引起的DNA甲基化对其印记基因缺陷性疾病的发生和后代生理功能的差异会有很大的影响。作者就冷冻保存技术对配子（精子和卵子）和胚胎的DNA甲基化的影响以及冷冻引起的DNA甲基化对后代的影响进行了综述,以期对配子的冷冻保存和辅助生殖技术的发展提供参考。     

哺乳动物胚胎玻璃化冷冻保存概况及其常用方法

哺乳动物卵母细胞和胚胎的超低温冷冻保存技术,是胚胎移植、体外受精、转基因和克隆等生物技术不可缺少的部[1].Rall和Fahy于1985年首次报道了用玻璃化方法来冷冻保存小鼠胚胎,这种方法不仅大大简化了冷冻过程,而且减少了由于细胞冰晶形成所引起的一系列物理及化学损伤,提高了冷冻效率[2].     

哺乳动物胚胎的冷冻保存

胚胎冷冻保存技术的应用使畜牧业的发展和实验动物的研究发生了很大的变化。本文主要介绍了哺乳动物胚胎冷冻保存的原理、方法、胚胎冷冻／解冻过程中存在的一些问题及其发展趋势。     

抗氧化剂在哺乳动物精液冷冻保存中的应用

从20世纪50年代哺乳动物精液冷冻研究开始,国内外研究者通过几十年对精液冷冻程序以及冷冻稀释液、解冻液等的筛选,牛、羊等家畜的冷冻精液已经商业化生产,广泛用于人工授精进行优良品种的繁殖.     

哺乳动物卵巢多肽调节因子及其生理意义

哺乳动物卵巢卵泡和卵母细胞的成熟和排卵是受卵巢内部和外部的许多化学信息控制的。卵巢外部的化学信息主要是指垂体前叶所分泌的促卵泡刺激(FSH)以及促黄体激素(LH);卵巢内部的化学信息包括人们早已熟悉的雌激素和孕酮这两种类固醇激素,近二十年来,人们发现卵巢除能分泌类固醇激素外,还能分泌某些非类固醇物质,这些类     

卵巢组织玻璃化冷冻保存的研究进展

就目前卵巢组织玻璃化冷冻保存的发展状况及影响卵巢冻融成功的因素等几个方面进行综述,并对卵巢组织冷冻保存所存在的问题及今后研究方向提出了自己的看法。     

The Effects of Sample Size on the Outcome of Ovarian Tissue Cryopreservation

Cryopreservation of ovarian tissue is known to affect follicular survival. Several variables may be responsible for this. Little attention has focused on the effect of the size of the fragment to be cryopreserved. This study was conducted to assess the effect of the size of the tissue on follicular histology after freezing with 1,2-propanediol. Histological evaluations were performed of control and cryopreserved tissue. Fragments were cut 10 × 3 × 2 mm³ (2 mm group) or 10 × 3 × 4 mm³ (4 mm group). Percentages of normal follicles in control fragments cut into 2 and 4 mm slices were 56% and 34%, respectively. The relative risks to obtain normal follicles in the 2 mm and the 4 mm fragments after cryopreservation were 0.63 and 0.47, respectively. Freezing reduced follicle survival to a significantly greater extent in the larger tissue fragments. There is an increased risk of damage to primary and primordial follicles, when the tissue slices are cut with all dimensions larger than 2 mm.     

The Use of an Open-freezing System with Self-seeding for Cryopreservation of Mouse Ovarian Tissue

Chemoradiotherapy in young women with cancer has substantially improved life expectancy in these patients, but these treatments often cause infertility. One method of preserving fertility is to cryopreserve ovarian tissue. In this study, an automatic open-vessel freezing system with self-seeding was tested for cryopreservation of murine ovarian tissue; the mouse is a species widely used in human and veterinary medical research. The freezing system concerned, is used for cryopreservation of oocytes and embryos in Europe. Twenty severe combined immunodeficiency (SCID) mice were ovariectomized. The ovarian tissue was either directly transplanted heterotopically into the neck muscle (group 1, n = 6) or cryopreserved after equilibration with 1.5 M dimethylsulphoxide and propanediol. After thawing, the tissue was transplanted in SCID mice (group 2, n = 6). Before and after thawing, a part of the ovarian tissue was examined with the LIVE/DEAD fluorescent viability staining. The count of follicles revealed intact (fresh 24.1%/thawed 21.7%), impaired (fresh 35.1%/thawed 35.4%), and dead follicles (fresh 40.8%/thawed 42.9%). The healthy follicular loss because of the cryopreservation was 10.0%. All recipient mice were killed after 3 weeks. Transplanted ovarian tissue was found macroscopically in all mice. Histological examination showed several growing follicles in all developmental phases in both groups of SCID mice [group 1 (fresh grafts): 315 +/- 76.3 (mean +/- SD); group 2 (cryopreserved grafts): 237 +/- 63.4]. These results demonstrate that the use of an open-freezing system allows the survival of cryopreserved mouse ovarian tissue.     

动物卵巢血管周期性变化及其与卵巢功能关系

血管新生是哺乳动物在出生后发生于周期性生理变化的器官中的特殊生理机制,卵巢作为具有明显周期性变化特征的器官,其上卵泡发育与闭锁、黄体的形成与退化均具有明显的血管周期性变化特点。在卵巢的周期性血管变化过程中,血管内皮生长因子(VEGF)和血管紧张素(Ang)起着重要的调控作用,其表达的时空特性与血管的周期性变化是密切相连的。     

牛乳腺组织的冷冻保存及乳腺细胞的分离

为研究牛乳腺组织冷冻保存的可行性,将牛乳腺组织剪成直径小于0.5 mm的组织块,以含20% NBS和10% DMSO的DMEM/F12作为冷冻保护液,按如下程序进行冷冻保存:4 ℃平衡1 h,－20 ℃平衡2 h,－40 ℃平衡2 h,－80 ℃保存。1周后,复苏牛乳腺组织,检测组织的存活率,分离培养乳腺细胞,检测细胞的存活率,观察细胞生长状态。结果显示,复苏后的牛乳腺组织可重新贴壁生长,贴壁存活率为62.5%,经消化离散,可分离培养出乳腺细胞,细胞存活率为58.5%,培养24 h后细胞存活率为46.3%。随着培养时间延长,细胞活力恢复,第1代细胞存活率为93.2%,生长状态良好。该乳腺组织的冷冻方法操作简单、方便、快捷,且很好地保持了乳腺细胞的活力,为稳定和增加乳腺细胞的来源提供了很好的途径,为进一步研究乳腺的结构和发育提供了素材。     

不同冷冻保护剂对犊牛睾丸组织冷冻效果的研究

通过探讨不同冷冻保护剂对犊牛睾丸组织冷冻保存的效果以及睾丸组织低损伤冷冻保存的机理,为保护珍稀濒危物种、恢复人类未成年男性生育能力等提供依据。研究将二甲基亚砜(DMSO)、甘油、丙二醇、蔗糖和葡萄籽原花青素(GSP)五种冷冻保护剂设置八个浓度梯度,用以进行犊牛睾丸组织的冷冻保存,7d后解冻,解冻后检测睾丸组织内的一氧化氮(NO)、一氧化氮合酶(NOS)和细胞活率。结果表明,经10%DMSO、丙二醇、蔗糖和GSP冷冻犊牛睾丸组织7d解冻后,组织细胞活率分别达到77%、62%、34%和24%,DMSO组优于其他组(P0.05);NO水平分别为0.52、0.51、0.64和0.49μmol/mgprot,NOS活性分别为1.03、0.98、1.04和0.96U/mgprot,GSP组优于其他组(P0.05)。7.5%甘油冷冻犊牛睾丸7d解冻后,细胞活率、NO和NOS分别为41%、0.54μmol/mgprot和1.07U/mgprot,优于组内其他浓度(P0.05)。表明10%DMSO能有效地冷冻保存犊牛睾丸组织。     

Effects of Cryoprotectant Agents on Equine Ovarian Biopsy Fragments in Preparation for Cryopreservation

The exposure effect of cryoprotectant agents (CPAs) on morphology of preantral follicles (PAFs), stromal cell and PAF densities, and area of equine ovarian fragments were evaluated. Three independent experiments with identical methodologies were performed. Each experiment was composed of one CPA (dimethyl sulfoxide, ethylene glycol, or propylene glycol) and was performed in three replicates. Ovarian biopsy fragments were harvested from six mares in each experiment and submitted to the cryoprotectants using four times of exposure (0, 10, 15, and 20 minutes). PAF and stromal cell densities, and area of the fragments were not affected (P > .05) by any of the CPAs throughout the time of exposure. However, the morphology of the PAFs was affected (P < .05) by the CPAs. In the propylene glycol and dimethyl sulfoxide, higher (P < .05) percentages of abnormal PAFs were observed at 10 and 20 minutes of exposure, respectively. The PAF morphology in the ethylene glycol treatments was not affected (P > .05) throughout the times of exposure. Positive correlations (r = 0.57–0.77; P < .001, power = 96%–99%) were identified between PAF density and stromal cell density in all experiments. In conclusion, (1) ethylene glycol seems to be a less harmful CPA to equine PAFs, (2) exposure to CPAs did not affect the cell density and area of ovarian fragments, (3) PAF density was positively correlated with stromal cell density, and (4) stromal cell density did not affect the morphology of PAFs.     

Technical Aspects of Laparoscopic Ovarian Autograft in Ewes After Cryopreservation by Slow-Cooling Protocol

Iatrogenic ovarian failure and infertility are long term‐term side effects of anticancerous gonadotoxic treatments in children or women of reproductive year. Ovarian cortex cryopreservation can be a solution to preserve immature germinal cells before gonadotoxic treatment, for later transplantation. The aim of our study was to prove the efficiency of a laparoscopic technique for orthotopic graft after a slow‐freezing/thawing protocol, and to evaluate the effect of ovarian cryopreservation and autograft on the primordial follicle survival rate. Experimental surgical study was performed on 6‐ to 12‐month‐old ewes. The study was approved by the ethic committee of the Lyon‐veterinary‐school. The left ovary was removed by laparoscopy and cut in half, and medulla was excised. In group 1 (n = 6), autograft was performed immediately on the right ovary, and in group 2 (n = 6), graft was performed after a slow‐freezing/thawing protocol. The second hemi‐ovary served as an ungrafted control fragment. A polypropylene/polyglactin mesh was included between graft and base to separate the two structures, to help histological analysis. The mean graft performance time was 71 ± 8 min in the first group and 57 ± 10 min in the second. Freezing did not affect the number of primordial follicles. In the ungraft control fragments, the global anomaly rate (cytoplasm plus nuclear anomaly) increased after freezing (p < 0.05). Others results did not reach signification. Pelvic adhesion occurred only once. The post‐graft primordial follicle survival rate was 5.1 ± 2.8% in the non‐frozen group vs. 6.3 ± 2.3% after freezing/thawing. Kruskal–Wallis and Wilkoxon non‐parametric tests were used for statistical analysis. Laparoscopy seems to be a well‐adapted technique for ovarian tissue orthotopic autograft. The main follicle loss occurs before graft revascularization. Our orthotopic graft’s procedure has to be improved to obtain a better graft’s neovascularization, and to have a better long‐term post‐graft primordial follicle survival rate.     

卵巢组织玻璃化冷冻研究进展

卵巢组织玻璃化冷冻可替代直接冷冻卵母细胞或胚胎。玻璃化冷冻卵巢组织在辅助生殖上具有优越性。它无需控制供体的生殖周期 ,也无需取出卵泡。同样这一技术可用于保存濒危动物或受意外伤害的人或动物的卵母细胞 ;可为性成熟前失去生殖能力的动物或人提供生殖保险以及增加卵母细胞的来源 ;可用于建立生殖细胞 (卵母细胞 )的冷冻库。而传统的冷冻技术存在很多弊端。文章综述了玻璃化冷冻卵巢组织的研究背景和现状 ,并指出了其广阔的应用前景