利用卵巢移植技术挽救濒危转基因小鼠方法的建立

目的:通过移植不孕不育转基因小鼠的卵巢到健康受体小鼠的相同位置,产生转基因小鼠的卵细胞,进一步交配获得转基因小鼠。方法:摘除健康受体小鼠卵巢,取出转基因小鼠卵巢,将转基因小鼠的卵巢组织移植入受体小鼠相同的位置。饲养15d后,与背景品系雄鼠交配,产生新生小鼠,剪尾鉴定,确定是否有阳性转基因小鼠出生。结果:移植的转基因雌鼠的卵巢可以在受体小鼠体内恢复功能,受体与背景品系雄鼠能正常交配,产生新生后代,进过基因鉴定,有转基因新生后代。结论:通过同位异体卵巢移植技术,挽救濒危转基因小鼠品系的方法可行。     

通俗遗传学——家蚕的人工生殖及其利用 Ⅲ生殖细胞的冷冻保存和精子的成熟

一、生殖细胞的冷冻保存本杂志笫二期(Vol.30,1991)中,叙述了有关家蚕的幼虫卵巢的冷冻保存,通过单性生殖回收到个体的试验。再举一个实验例子,来看一看新保(1989)的实验结果,即把幼虫的卵巢及精巢于冰点以下196℃的液态氮中冷冻保存,融解后将它们移植到同龄幼虫,使移植的卵巢或精巢与各宿主的导管接合,从而回收到个体。基本冷冻介液中的甘油     

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.     

受体山羊卵巢有无卵泡对移植妊娠率的影响

为了提高受体山羊移植妊娠率,试验在生产条件下对111只波尔山羊供体实施超数排卵处理,子宫生产胚胎,然后对977只奶山羊受体分两组实施胚胎移植,一组卵巢上只有黄体,另一组卵巢上除有黄体外,还有卵泡(卵泡较小,一般小于黄体),并对移植妊娠率进行比较分析。结果表明:有卵泡受体移植后妊娠率为52.65%;无卵泡受体移植后妊娠率为60.13%,差异显著(P<0.05),说明选择卵巢上无卵泡的受体进行移植妊娠率较高。卵巢上有卵泡的受体妊娠率为52.65%,在生产上也属于理想效果。     

卵巢异种移植在生殖生物学和动物保存中的研究进展

随着关于卵巢组织较有效的冷冻保存方法的发展,生殖系的保存和繁殖已变成了现实的目标。本文就卵巢冷冻保存和卵巢移植上的发展进步,异种移植在保存稀有和濒危物种及一些女性卵巢功能上的潜在价值,以及即将出现的异种移植技术进行综述。     

植物甾醇对去卵巢KM小鼠血清生殖激素及乳腺组织孕激素受体的影响

本试验旨在研究植物甾醇对去卵巢小鼠血清激素及乳腺组织中孕激素受体的影响。50只雌性小鼠,随机选40只进行去卵巢手术,恢复7 d。分组如下：正常组、去卵巢对照组、植物甾醇低、中、高3个剂量组（每日灌胃20、80和320 mg/kg植物甾醇）。连续灌胃3周,第22天采集血样和乳腺组织,对小鼠血清中的雌二醇、孕酮和催乳素水平进行检测,对乳腺组织的孕激素受体基因表达进行荧光定量检测。结果表明,去卵巢对照组小鼠雌二醇水平显著低于正常组（P〈0.05）。植物甾醇组小鼠血清雌二醇水平持续升高,其中,高剂量组小鼠血清雌二醇水平与正常组最接近;与去卵巢对照组相比,去卵巢植物甾醇组小鼠血清孕酮水平均有提高,但差异不显著（P〉0.05）;与去卵巢对照组相比,去卵巢植物甾醇组小鼠催乳素水平先下降后上升,但他们之间无显著差异（P〉0.05）。与去卵巢对照组相比,植物甾醇各剂量组小鼠孕激素受体基因相对表达量呈上升趋势,低、中、高剂量组小鼠孕激素受体基因相对表达量分别是去卵巢对照组的1.05、1.13和2.78倍。植物甾醇可改善去卵巢小鼠内源激素不平衡的状况,促进乳腺组织PR基因的表达。     

全反式视黄酸对小鼠卵泡发育、繁殖性能和生殖激素水平的影响

本研究旨在探讨全反式视黄酸（ATRA）对昆明小鼠卵泡发育、繁殖性能和生殖激素水平的影响。试验选取7周龄雌性昆明小鼠24只,适应性饲养5～7 d后,随机分为4组,每组6个重复,每个重复1只,分别为对照组（灭菌蒸馏水）、低剂量组（0.1 mg/kg ATRA）、中剂量组（1.0 mg/kg ATRA）和高剂量组（10.0 mg/kg ATRA）,连续灌胃14 d。各组分别取6只昆明小鼠采样,测定血清雌激素（E2）、孕酮（P4）和促卵泡生成素（FSH）水平,同时分离卵巢并计算卵巢系数,制作卵巢组织石蜡切片,并进行卵泡计数。选择未发情的昆明小鼠64只,随机分为4组,每组16个重复,每个重复1只。剂量同上。连续灌胃ATRA 1个发情周期（5 d）。将发情小鼠各组取6只采样,测定血清激素水平,其余10只与雄性小鼠合笼,妊娠期间每周称量1次体重,分娩之后统计窝产仔数、出生窝重与出生个体重。结果表明：1）昆明小鼠连续灌胃ATRA14 d,与对照组相比,ATRA对小鼠的体重与卵巢系数均没有显著影响（P>0.05）。2）与对照组相比,高剂量组初级卵泡的数量显著提高（P<0.05）,而闭锁卵泡数...     

受体和移植时间对小鼠睾丸移植效果的影响

通过比较不同受体和不同移植时间对睾丸移植效果的影响,以期探索建立疾病模型小鼠安全保存的新方法。按受体不同分为C57BL/6J受体8周组和裸鼠受体8周组,按移植时间的不同分为裸鼠受体8周组和裸鼠受体10周组。移植后处死受体鼠回收移植物。测试和分析移植物存活率、回收物增重的倍数和生殖细胞的分化情况。结果表明:不同试验组移植睾丸的生殖细胞分化差异显著,裸鼠受体8周组的分化情况好于C57BL/6J受体8周组(P<0.01);裸鼠受体10周组分化好于裸鼠受体8周组(P<0.05)。由此可见裸鼠受体的移植效果要好于同品系受体,将睾丸移植时间从8周延长到10周有利于生殖细胞的分化。     

利用体外受精-胚胎移植技术进行ALK3基因敲除小鼠的保种鉴定

建立用于ALK3基因敲除小鼠品种保存的体外受精一胚胎移植(IVF-ET)模型.采用杂合子雌性ALK3小鼠卵子与杂合子雄性ALK3小鼠精子进行体外受精,收集2-cell,进行胚胎冷冻,1周后进行胚胎复苏移植,通过PCR方法进行子代鉴定.结果表明,冻存胚胎320枚,复苏获得存活胚胎96枚,移植80枚,产子23只,获得杂合子11只,通过体外受精一胚胎移植可对ALK3基因敲除小鼠进行保种鉴定.     

胚胎冷冻保存研究进展(综述)

自从 Polgy、Wmith 和 Porkes 首次发现冷冻保存精子的方法后,就引起了繁殖工作者冷冻保存哺乳动物卵子和胚胎的兴趣。1972年 Whittmghan 首次报道鼠胚胎冷冻成功,在-196℃保存小鼠胚胎移入受体有29%产仔。不久,Wilmut 也报道了鼠胚胎冷冻成功。1973年 Wilnnut 和 Rosuson 第一次报道冷冻牛胚胎得到一头犊牛。从此,冷冻保存哺乳动物胚胎的研究才不断开展。开始,对实验室动物研究较多,后来转到了家畜方面,迄今已用冷冻保     

卵巢移植技术及其应用研究进展

近20年来,卵巢移植作为重要的辅助生殖技术已取得显著研究进展,相继成功地开展了卵巢自体原位移植,自体异位移植,同种异体移植和异种移植.目前利用新鲜卵巢或冷冻保存卵巢进行移植,已出生多例卵巢移植的新生个体.这表明卵巢移植技术与冷冻保存技术相结合,可成为保存和挽救雌性个体生殖能力的一种重要手段,尤其在稀有濒危动物中具有特殊...     

Cryopreservation of canine ovaries by vitrification

The cryopreservation of ovarian tissues is a technology with significant potential for the preservation of the genetic resource materials of working dogs, including guide dogs for the blind. However, no attempt has been reported on cryopreservation of the canine ovary. Thus, we evaluated a vitrification method for cryopreservation of canine ovaries and determined the potential functionality of vitrified-warmed canine ovaries by means of transplantation into non-obese diabetic-severe combined immunodeficiency (NOD-SCID) mice. All ovarian tissues cryopreserved by vitrification were morphologically normal in terms of histology. Cryopreserved ovaries were transplanted into the ovarian bursa of the NOD-SCID mice, and the xenografts were recovered from 23 of 23 mice (100%) 4 weeks after the operation. The transplanted canine tissue was tightly adhered to the mouse ovary. Although antral follicle formation did not occur after grafting, proliferating cell nuclear antigen immunoreactivity was detectable in many of the granulosa cells in the primary follicles of the grafts. These results indicate that cryopreservation of the canine ovary by vitrification appears to have the potential to restore endocrine function and ovulation potential.     

卵巢玻璃化冷冻保存研究进展

卵巢玻璃化冷冻是一种操作简单、冷冻效果好的卵巢组织冷冻降温技术,对动物繁殖能力保存、物种保护、生物多样性保存,以及动物胚胎工程和人类临床医学研究与应用等具有重要意义。卵巢组织冷冻保存较卵母细胞冷冻保存,具有明显的优点。在冷冻保护剂的选择、冷冻材料、冷冻平衡及冷冻方法上都有其特殊之处。卵巢组织冷冻保存技术已在人类医学临床上发挥其应用价值。     

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。     

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     

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.     

Irreversible damage in ovine ovarian tissue after cryopreservation in propanediol: analyses after in vitro culture and xenotransplantation

Current progress in cancer treatment has increased the incidence of long-term patient survival. Ovarian tissue cryopreservation (OT) is still the most promising fertility saving method offered to young female patients with cancer prior to the onset of radio-chemotherapy. Further follicular development of immature primordial follicles depends on transplantation or in vitro culture (IVC). Aim of this study was to evaluate the appropriateness of cryopreserved ovine OT with 1,2-propanediol (PROH) after short-term IVC and xenotransplantation (XT). Ovarian tissue fragments from young adult sheep were cryopreserved using a standard slow-freezing protocol with 1.5 M PROH. Cryopreserved OT was assessed by light- and transmission electron microscopic analyses after thawing, IVC or XT in severe immunodeficient mice. Control OT showed the presence of healthy preantral follicles (Mean: 78.8%; SE 2.9%) and normal structure of the stromal tissue. After thawing and IVC over 80% of damaged primordial follicles and poor preservation of the stromal tissue was observed. After XT, OT demonstrated deficient follicles and huge areas of vacuolization in the stromal tissue confirmed by ultrastructural assessment. In conclusion, because of the irreversible character of the follicular and stromal damage of cryopreserved ovine ovarian tissue after IVC and XT, strong improvement of the utilized protocol is needed to be suitable for the preservation of ovine ovarian tissue. The deleterious effects of PROH do not imply its exclusion as cryoprotectant, but more research is needed for the development of less toxic cryoprotectant mixtures and toxicity neutralizers with attested cryoprotectant capacity for the safe and feasible freezing of human ovarian tissue.     

Effect of vitrification of feline ovarian cortex on follicular and oocyte quality and competence

Cryopreservation of ovarian cortex has important implications in the preservation of fertility and biodiversity in animal species. Slow freezing of cat ovarian tissue resulted in the preservation of follicular morphology and in the follicular development after xenografting. Vitrification has been recently applied to ovarian tissues of different species, but no information is available on the effect of this method on feline ovarian cortex. Moreover, meiotic competence of fully grown oocytes isolated from cryopreserved tissue has not been reported. The aim of this study was to evaluate the effect of vitrification of feline ovarian cortex on follicular morphology and oocyte integrity, as well as meiotic competence. A total of 352 fragments (1.5-2 mm(3) ) were obtained from ovarian cortical tissues: 176 were vitrified and 176 were used fresh as control. Histological evaluation of fresh and vitrified fragments showed intact follicles after cryopreservation procedures with no statistically significant destructive effect from primordial to antral follicles. After IVM, oocytes collected from vitrified ovarian fragment showed a higher proportion of gametes arrested at germinal vesicle (GV) stage compared to those isolated from fresh control tissue (33.8% vs 2.9%; p < 0.001). However, oocytes isolated from vitrified tissues were able to resume meiosis, albeit at lower rate than those collected from fresh tissues (39.8% vs 85.9%; p < 0.00001). Vitrification induced changes in the organization of cytoskeletal elements (actin microfilaments and microtubules) of oocytes, but significantly only for actin network (p < 0.001). Finally, chromatin configuration within the GV was not affected by the cryopreservation procedure. Our study demonstrated that vitrification preserves the integrity of ovarian follicles and that oocytes retrieved from cryopreserved tissue maintain the capability of resuming meiosis. To our knowledge, this has not previously been reported in the cat.     

Simple prediction of the survival of follicles in cryopreserved human ovarian tissue

This study examines the possible predictive value of the LIVE/DEAD fluorescence viability assay for evaluation of survival of cryopreserved human ovarian tissue. Ovarian tissue from ten patients was examined by LIVE/DEAD viability staining before and after cryopreservation and after freezing in a -20 C freezer (negative control). After cryopreservation with a slow freezing protocol and cryoprotectant the LIVE/DEAD assay showed 86% viable follicles (an intact oocyte and at least more than 50% of the granulosa cells alive), whereas after freezing at -20 C the survival rate was 67%. The healthy follicular loss after cryopreservation was 4%, whereas with freezing at -20 C, it was 25%. Although this assay overestimates the survival rate of cryopreserved primordial follicles, if the LIVE/DEAD assay yields greater than approximately 85% viable follicles, it can be assumed that the follicles in the cryopreserved tissue have maintained their developmental potential and that the tissue is suitable for retransplantation.