猪腔前卵泡体外培养研究进展

哺乳动物卵巢中有数量丰富的腔前卵泡,腔前卵泡体外发育的研究,对于揭示卵子发生和卵泡发育的内在规律有重要意义,并可以最大限度利用卵巢资源促进动物繁殖,保护濒临灭绝物种及人类生殖健康。猪腔前卵泡自开始研究以来,已取得了很大的进展。作者简要阐述了猪腔前卵泡培养方法、培养条件的研究进展及其体外培养技术存在的问题和发展前景。     

体外培养牛腔前卵泡颗粒细胞的超微结构研究

透射电镜下，简易机械法分离得到的腔前卵泡有1～3层颗粒细胞，相邻颗粒细胞间存在明显而广泛的间隙连接，卵泡基膜完整，外无卵泡膜。培养过程中超微结构的变化与体内发育腔前卵泡类似。培养6d时观察到颗粒细胞增殖现象，培养15d时，个别卵泡的内膜细胞开始形成。超微结构研究结果表明，本培养体系适于小腔前卵泡的体外培养。     

小鼠腔前卵泡分离方法的研究

<正>哺乳动物卵巢皮质中含有大量的腔前卵泡,但90%在发育过程中会不断闭锁退化。开发和利用这一潜在的种质资源具有重要的实用价值,因此对腔前卵泡进行体外培养显得尤为重要,有效地将腔前卵泡分离出来是进行体外培养的第一步。虽然有关啮齿类动物的分离体系研究已取得了一定进展,但由于各试验条件的不同,分离体系仍存在许多问题,由于家畜卵巢组织的结构致密,纤维化程度比较高,问题更突出。因此,许多学者对腔前卵泡的分离进行了长期     

牛腔前卵泡体外培养前后质量评定

采用台盼蓝染色、相差显微镜观察以及透射电镜方法对体外培养前后牛腔前卵泡活力进行评定。结果表明,台盼蓝染色和相差显微镜观察检测卵泡活力有一定的误检率,超微结构检测能够客观、真实地反映腔前卵泡健康状况,可作为评定腔前卵泡培养系统优劣的一个可靠手段。在实际应用中,相差显微镜观察与超微结构评定相结合可发挥良好作用。     

哺乳动物卵泡发育过程中的超微结构变化

目前 ,动物腔前卵泡的体外培养正日益受到重视 ,并已取得了较大进展 ,已建立的培养体系可成功地使腔前卵泡发育到有腔阶段 ,猪、山羊等已可实现腔前卵泡卵母细胞体外成熟 ,体外受精并发育至囊胚阶段。但有关腔前卵泡体外成熟的机制仍不明了。本文通过对体内发育与体外培养之卵泡及其卵母细胞超微结构进行比较 ,从微细结构上客观评定体外培养卵泡的形态、活力、代谢状况及功能完整性 ,界定体外生长卵泡所处发育阶段 ,从而为确立和完善腔前卵泡体外培养体系并最终选择高质量的卵母细胞进行体外受精提供可靠的理论依据。1　体内发育卵泡及其卵…     

小鼠腔前卵泡体外培养研究进展

哺乳动物卵巢中有数量丰富的腔前卵泡,小鼠腔前卵泡卵母细胞从开始尝试分离至今已经获得试管后代,为在动物生产中的应用奠定了良好的理论基础和技术路线。文章简要地综述了小鼠腔前卵泡体外培养的方法,主要讨论了血清、生殖激素等培养液添加成分对小鼠腔前卵泡培养的影响及小鼠腔前卵泡体外培养技术的发展前景。     

哺乳动物腔前卵泡卵母细胞的体外培养

阐明哺乳动物卵泡体内发生的一般模式，回顾腔前卵泡体外培养的研究历史，着重论述腔前卵泡的获取及鉴定标准，常用的培养体系和影响腔前卵泡体外发育的多种因素。     

牛腔前卵泡在体外无血清培养中发育为有腔卵泡

用无血清培养系统研究了牛腔前卵泡的体外培养。直径为100－200μm的牛腔前卵泡在添加L－谷氨酰胺、BSA、睾酮、转铁蛋白和硒的McCoy′s 5a培养液中培养，卵泡保持正常的形态结构并持续生长，培养10d左右形成贸泡腔，成腔率约50％。培养液中添加胰岛素对卵泡直径的增长和卵泡腔的形成有明显的促进作用，但添加FSH对腔前卵泡的生长未表现促进作用。     

猪腔前卵泡机械分离研究

本研究以分离到的卵泡数量、分离时间和卵泡在体外培养3 d后的存活率为指标比较了两种机械方法分离猪腔前卵泡的效果。结果表明:用针刮法分离猪腔前卵泡的数量(Φ<50μm:203600±59000;50μm≤Φ≤150μm:29.40±10.34;Φ>150μm:9.30±3.29)极显著(P<0.01)高于剪碎法(Φ<50μm:18900±12000;50μm≤Φ≤150μm:13.95±3.70;Φ>150μm:4.95±1.61),而且针刮法16.16±1.43 min分离时间比剪碎法(20.30±1.48)min短,(P<0.01)。体外培养3 d后,腔前卵泡存活率在两种方法之间没有明显差异。说明应用针刮法分离猪腔前卵泡能有效保护卵母细胞和颗粒细胞的正常形态以及基膜的完整性,从而使分离到的猪腔前卵泡维持正常的生理活性。     

哺乳动物腔前卵泡体外培养及研究进展

哺乳动物卵巢内的原始卵泡库是雌性生殖资源的储存库,但大部分卵泡在腔前阶段已退化闭锁了,这无疑是对这一资源的巨大浪费。因此,越来越多的学者致力于腔前卵泡的开发和利用,建立了一系列的培养体系,尤其在小鼠上做的比较成功,已经得到了少量后代。文章主要就卵泡的发生发育过程,腔前卵泡的分离方法,培养基的选择,不同直径腔前卵泡培养体系的建立,影响腔前卵泡发育的因素及研究进展进行了阐述,为进一步完善腔前卵泡培养体系,揭示其发育机理提供参考。     

卵巢大小及发育状况与牛腔前卵泡采集数量的关系

用简单机械分离法处理了 12 7枚成年牛卵巢。结果显示 ,在外观正常的卵巢中 ,腔前卵泡的采集数量与卵巢的大小成正相关关系 ,而有无黄体与腔前卵泡的采集数量无明显关系 ;卵巢上不同大小的可见卵泡的数量和分布与腔前卵泡的采集量有关。卵巢上可见卵泡分布均衡 ,大、中、小卵泡均有分布 ,小卵泡不过多以及无大卵泡 ,但中、小卵泡较多的 ,无论是否有黄体存在 ,均可获得较多腔前卵泡。而卵巢表面脂肪化、卵巢充血、有弥散性片状黄体及幼稚卵巢的 ,则腔前卵泡分离很少或几乎分离不到     

Evaluation of in vitro culture systems for goat preantral follicles using reused ovaries from reproductive biotechniques: An alternative to maximize the potential of reproduction

This study aimed to examine the in vitro culture of secondary preantral follicles, using reused ovaries, to compare both the 2D and 3D methods of in vitro culture of preantral follicles, and the system of medium replacement. Twenty‐five pairs of ovaries from mixed‐breed goats were used for the experiment. Follicular puncture of antral follicles was performed for in vitro production. After this procedure, the secondary preantral follicles were submitted to a microdissection procedure. The isolated preantral follicles were randomly divided into three treatments: (a) Two‐dimensional culture with partial replacement of medium during culture (2D PR), (b) Three‐dimensional culture with addition of medium during culture (3D AD) and (c) Three‐dimensional culture with partial replacement of medium (3D PR). The culture period was 18 days. All treatments at the end of the in vitro culture period (18 days) presented a follicular survival rate which ranged from 59% to 70%, demonstrating that it was possible to perform an experiment with preantral follicles using ovaries that had previously been used in another reproductive biotechnique. The 3D AD treatment showed a survival percentage and follicular diameter higher than the 2D PR treatment, however, it did not differ from the 3D PR treatment. In conclusion, experiments employing the use of preantral follicles can be performed with success after the ovaries have been used for experiments with antral follicles. Moreover, the three‐dimensional system with the addition of medium is recommended for in vitro culture of preantral follicles, since this system is more practical and financially feasible.     

Gene expression and immunolocalization of fibroblast growth factor 2 in the ovary and its effect on the in vitro culture of caprine preantral ovarian follicles

This study quantified Fibroblast growth factor 2 (FGF-2) mRNA and localized FGF-2 protein in different categories of follicles isolated from goat ovaries. In addition, we verified the effects of this factor on the in vitro culture of preantral follicles isolated from goats. For mRNA quantification, we performed real-time PCR using primordial, primary and secondary follicles, as well as cumulus-oocyte complexes (COCs) and mural granulosa and theca cells of small and large antral follicles. For FGF-2 protein localization, the ovaries were subjected to conventional immunohistochemical procedures. Preantral follicles were isolated and cultured in vitro for 12 days in either control (basic) or supplemented with FGF-2 medium. The expression of FGF-2 mRNA was detected in all categories of follicles and there was no difference in preantral follicles and COCs or granulosa/theca cells from small and large antral follicles. However, in large antral follicles, COCs showed expression levels significantly lower than in granulosa/theca cells (p < 0.05). We observed moderate expression of FGF-2 protein in preantral follicles but not in granulosa cells of primordial follicles and theca cells of secondary follicles. In both small and large antral follicles, strong, moderate and weak staining was observed in oocytes, granulosa and theca cells, respectively. The addition of FGF-2 caused a significant increase in the daily follicular growth rate compared to the control group. We conclude that FGF-2 mRNA is expressed throughout follicular development and that its protein can be found in different patterns in preantral and antral follicles. Furthermore, FGF-2 increases the follicular growth rate in vitro.     

Vitrification of mouse preantral follicles versus slow freezing: Morphological and apoptosis evaluation

The aim of this study was evaluation of survivability, maturation rate and apoptotic gene expression of preantral follicles after vitrification and slow freezing technique. Normal mouse preantral follicles were randomly divided into three experimental groups. In the control group, follicles were cultured immediately; in the vitrification and slow freezing groups, follicles were cultured after vitrification‐warming and slow freezing‐thawing procedures. Follicular viability was assessed by using 0.4% trypan blue, and molecular evaluation of messenger RNA levels of apoptosis‐related genes was performed by the semi‐quantitative RT‐PCR method after 3 h of culture. Oocyte maturation rates were also evaluated on day 14 of culture. Survival and maturation rate in the slow freezing group were significantly lower than those in control and vitrification groups (P ≤ 0.05). Although there was no difference in Survivin expression among the three experimental groups, Bcl‐2 expression was significantly lower in the slow freezing group compared to the other groups (P ≤ 0.05). The expression of Bax, P53, Fas and Bax/Bcl‐2 ratio in the slow freezing group was significantly higher than control and vitrification groups (P ≤ 0.05). Preantral follicle vitrification seems to be better than slow freezing as seen in the survival, maturation and expression rates of apoptotic gene variants.     

牛卵巢黄体状况与腔前卵泡采集数量的关系

根据牛离体卵巢黄体的不同状况，将31枚卵巢分为5种类型，并采用机构方法分离腔前卵泡，观察不同黄体状况卵巢与腔前卵泡采集数量的关系，结果表明，火山口型，圆锥型和蘑菇型3种大黄体的卵巢腔前卵泡采集量多，扁平片状和表面无黄体型卵巢腔前卵泡采集量较少，具有黄体状况的卵巢初级卵泡（Pm)采集数量多于无黄体类型卵巢，原始卵泡（Pf) 以3种黄体较大的卵巢采集数量最多，次级卵泡（Sc)则以黄体为火山口型卵巢为最多，说明卵巢黄体状况不同，其腔前卵泡采集数量有所不同。     

Methods for Equine Preantral Follicles Isolation: Quantitative Aspects

The aim of this study was to test the use of mechanical and mechanical‐enzymatic methods, saline solution (SS), and PBS solution for the manipulation and isolation of mare ovarian preantral follicles (PAFs). The ovaries were subjected to mechanical isolation (mixer) alone or in association with enzymatic digestion (collagenase). Incubation times of 10 and 20 min were employed. In the first group, 4.1 ± 4.9 PAFs were harvested with the mechanical‐enzymatic method vs 71.1 ± 19.2 with the mechanical procedure, showing a significant difference between methods; using SS and PBS, these numbers were 35.7 ± 34.3 and 39.6 ± 39.6, respectively, with no significant difference between solutions. In the second group, there was significant difference between methods, with 7.1 ± 10.6 follicles harvested with the mechanical‐enzymatic method vs 63.2 ± 22.9 with the mechanical procedure; using SS and PBS, means were 35.5 ± 36.4 and 34.9 ± 31.1, respectively. The mechanical method proved more effective than the mechanical‐enzymatic approach. Both SS and PBS can be used as a media for equine PAFs preparation.     

哺乳动物腔前卵泡的分离与培养

哺乳动物卵巢中绝大多数卵母细胞以无腔形式存在，有腔卵泡所占比例很少。通过建立腔前卵泡的培养体系，获取大量的具有成熟和受精能力的卵母细胞，将极大地促进体外受精、核移植等胚胎工程技术的发展，并有利于研究卵泡和卵母细胞的发育规律。