山羊卵巢无腔卵泡卵母细胞的体外生长

山羊卵巢无腔卵泡卵母细胞在以ＤＭＥＭ为基础,添加ＨＥＰＥＳ（２０ｍｍｏｌ／Ｌ）、ＦＣＳ（１０％）、ＦＳＨ（４０ｍｇ／Ｌ）、次黄嘌呤（２ｍｍｏｌ／Ｌ）、异双丁酰环腺苷酸（２ｍｍｏｌ／Ｌ）、ＩＧＦ－Ｉ（５０μｇ／Ｌ）、氢化可的松（４０μｇ／Ｌ）和ＩＴＳ（５０μｇ／Ｌ）的培养液中得以存活并生长。在二维培养体系中,卵泡在体外的生长模式和体内有很大差别。最显著的特征是卵泡不像在体内那样保持完整的立体结构一直到结束。绝大部分卵泡不同程度地发生基膜溶解和破裂,颗粒细胞向四周扩展并贴壁,形成单层。由于卵泡原有三维立体结构的破坏,易于导致卵母细胞的迁移。在生长方式上以数个卵泡聚集生长对其卵母细胞的生长似乎较为有利。卵泡卵母细胞在体外培养９ｄ,其直径可达１５０μｍ以上,达到了成熟时体积,存活率为５３％。实验证明,山羊无腔卵泡卵母细胞在合适的培养体系中,能在体外存活并生长,并能发育到成熟时的大小。     

生长因子对山羊无腔卵泡卵母细胞体外生长的影响

山羊无腔卵泡卵母细胞在胰岛素样生长因子I（IGF－I）和表皮生长因子（EGF）及碱性成纤维细胞生长因子（bFGF)单独或联合存在时，与FSH－道体外培养9d，结果表明，IGF－I（100ng/mL)能有效地维持卵泡卵母细胞的存活并促进其生长，存活率达75.00％，相对生长率达45.06％；EGF（50ng/mL)能提高卵泡卵母细胞的体外存活率，类似于IGF－I，但对其生长相对表现出抑制影响；bFGF(50ng/mL)对卵泡卵母细胞的体外存活具有促进作用，但对卵母细胞生长的促进作用不明显（卵母细胞的存活率为76.92%,相对生长率为31.55％）。IGF－I（100ng/mL)和EGF（50ng/mL)联合存在时，不论是对卵泡卵母细胞的存活，还是对生长均产生了最好的效果，卵母细胞存活率达到90.91％，相对生长达48.24％。本试验结果一个共同点是在FSH存在的情况下，这3种生长因子单独或有选择的联合，对山羊卵泡卵母细胞的体外存活能力均具有正向调节作用。     

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…     

卵泡数量对陕北白绒山羊卵母细胞体外成熟与孤雌发育的影响

为了进一步探索陕北白绒山羊卵母细胞体外成熟的方法,试验以屠宰场陕北绒山羊卵巢为材料,采用抽吸法收集直径大于2 mm卵泡的卵母细胞,研究卵泡数量对卵母细胞体外成熟和孤雌发育的影响。根据卵泡数目的差异分为3组,其中卵泡数目大于15个的设为Ⅰ组,卵泡数目在8~14个之间的和卵泡数目在1~7个之间的分别设为Ⅱ组和Ⅲ组。对山羊卵母细胞及孤雌激活后胚胎进行体外培养后,进而根据山羊卵母细胞体外成熟率、孤雌激活胚卵裂率和囊胚发育率来分析卵泡数量对其发育的影响。结果显示卵泡数目在8~14个之间的Ⅱ组山羊卵母细胞体外成熟率显著高于其他两组(P0.05),而这3组间的卵裂率和囊胚率差异不显著(P0.05)。表明山羊卵泡数目的过多或过少降低了卵母细胞体外成熟效率,且对孤雌激活胚的发育能力无影响。     

山羊卵泡颗粒细胞和输卵管上皮细胞共培养对山羊体外受精胚胎发育的影响

试验共分 3组 :对照组为单独培养液 ;第 2组为单层颗粒细胞 ;第 3组为输卵管上皮细胞 ,将山羊体外受精胚胎随机放入以上 3组处理中。结果显示山羊体外受精胚胎在卵泡颗粒细胞中发育到囊胚的比率为 4 1.0 % ,在输卵管上皮细胞中发育到囊胚的比率为 5 8.1% ,而在M199培养液中为 5 .6 %。山羊体外受精胚胎分别与山羊卵泡颗粒细胞和输卵管上皮细胞共同培养时 ,均可提高山羊体外受精胚胎的发育进程。且输卵管上皮细胞比颗粒细胞能显著提高山羊体外受精胚胎的发育进程 (P <0 .0 5 )。     

维生素对牛腔前卵泡体外培养的影响

用改良的McCoy’s 5a无血清培养液 (含 3mML -谷氨酰胺、0 1%BSA、2 0ng/ml睾酮、2 5 μg/ml转铁蛋白、10 0ng/ml胰岛素和 4ng/ml硒 ) ,在 96孔培养板中 ,每孔 2 5 0 μl培养液的条件下 ,研究了维生素C和维生素E对腔前卵泡体外发育的影响。结果表明 :在培养液中添加 90 μM的维生素C有利于维持腔前卵泡体外生长时结构的完整性 ,显著提高牛腔前卵泡体外培养 10天的存活率 (P <0 0 5 ) ,但并没有发现对腔前卵泡的生长、发育和成腔有促进作用 (P >0 0 5 ) ;在培养液中添加3 0 μM的维生素E对Φ >13 0 μm的腔前卵泡及其卵母细胞有促进发育和促进卵泡腔形成的趋势 (P >0 0 5 ) ;在培养液中联合添加 12 μM维生素E和 96μM维生素C ,能显著提高牛腔前卵泡体外培养 10天的存活率 (P <0 0 5 ) ,并对牛腔前卵泡的生长和发育有一定的促进作用 ,但差异不显著 (P >0 0 5 )。     

BMP-6对猪卵母细胞体外成熟的影响

为研究BMP-6在猪卵泡发育过程中的作用,采用免疫荧光和Western blot技术检测BMP-6在猪排卵前卵泡卵母细胞、颗粒细胞以及卵泡液中的表达;在猪卵母细胞体外成熟培养过程中分别添加5、25及50 ng/mL BMP-6蛋白,观察其对猪卵母细胞在体外成熟过程中的影响。结果表明,猪排卵前卵泡的卵母细胞和颗粒细胞以及卵泡液中均存在BMP-6蛋白,体外添加不同浓度的BMP-6蛋白均能明显促进猪卵母细胞体外成熟,说明BMP-6在猪卵泡发育及猪卵母细胞的成熟过程中发挥作用。     

小尾寒羊腔前卵泡体外生长发育的研究

近20年来,胚胎移植、体外受精、核移植、转基因等胚胎工程技术在理论和生产应用上已取得了很大进展.然而,所有这些技术是建立在具有完全发育能力的卵母细胞的基础之上的.大量卵母细胞的体外成熟培养是这些技术的限速步骤之一.目前采用的有腔卵泡卵母细胞的体外成熟和超数排卵,不能提供充足的卵母细胞以支撑胚胎工程技术的进一步发展.此外,卵巢中的卵母细胞绝大多数以无腔的形式存在于卵巢皮质内,有腔卵泡所占比例不到1%,屠宰场屠宰羊只后的卵巢内含有大量的腔前卵泡.如果建立腔前卵泡的体外培养体系,获得大量的具有成熟和受精能力的卵母细胞,一方面将会最大限度地挖掘保存卵巢上遗传资源;另一方面将极大的促进胚胎工程技术的研究与应用,还有利于研究卵泡和卵母细胞的生长和发育规律.     

动态添加FSH对绵羊卵巢皮质组织体外培养的影响

本研究旨在探索促卵泡素(FSH)对体外培养绵羊卵巢原始卵泡和组织活性的影响。通过稳定或动态方式添加不同浓度FSH(1、10、50、100ng·mL-1),用组织学和免疫组化PCNA方法来评估FSH对体外培养绵羊卵巢皮质组织中原始卵泡激活、生长、存活以及颗粒细胞增殖活性的影响;此外通过测定培养液中雌激素含量,评估卵巢组织体外培养过程中活性的保持。结果表明,先升后降动态添加FSH对于卵泡发育、存活和生长以及颗粒细胞的增殖具有全面的促进作用;同时组织分泌雌激素能力也高于其他组。以稳定方式添加时,50ng·mL-1FSH在培养1d时显示对卵泡发育的显著促进作用(P0.05),添加10ng·mL-1以上浓度的FSH可以促进卵泡存活和生长(P0.05)。在整个培养期内,各培养组中雌激素产量持续增加,说明卵巢活性在本培养体系中可以得到很好的维持。本试验结果表明FSH可能参与早期卵泡的发生,动态添加FSH更有利于体外培养卵泡的发育和组织活性的维持。     

哺乳动物卵泡发育过程中的超微结构研究进展

通过对体内发育与体外培养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提供可靠的理论依据。     

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.     

The effect of IGF-1 and FSH on the in vitro development of caprine secondary follicles and on the IGF-1, IGFR-I and FSHR mRNA levels

This study verified the in vitro effects of IGF-1, FSH or both on caprine preantral follicle development and mRNA levels encoding IGF-1, IGFR-1 and FSHR. Secondary follicles were cultured for six days with FSH, IGF-1 or IGF-1+FSH. The results showed that IGF-1 and/or FSH addition did not influence follicular development for six days. The interaction between IGF-1 and FSH increased the mRNA levels of IGF-1 and FSHR, and FSH increased the expression of the IGFR-1 mRNA. Thus, IGF-1 and/or FSH increased IGF-1, IGFR-1 and FSHR mRNA levels in in vitro cultured caprine secondary follicles, but they did not influence their development after six days of in vitro culture.     

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.     

JSAR Outstanding Research Award. In vitro growth of mammalian oocytes

The mammalian ovary contains a huge number of small follicles of various sizes, and each follicle encloses a small oocyte. Only a small number of non-growing oocytes (30 microm in the pig and cow) grow to their final size (120 microm), mature, and are ovulated. In vitro growth (IVG) culturing of small oocytes will provide a new source of mature oocytes for livestock production. Using the IVG culture system, non-growing mouse oocytes in primordial follicles grow to their final size and acquire full developmental competence. Among large animals, babies were produced from ovarian oocytes by IVG culture only in the cow. However, the oocytes used were not non-growing ones but at the mid-growth stage (90-99 microm in diameter) in early antral follicles. Xenotransplantation of the follicles at an early stage to immuno-deficient mice is a substitute for an effective long-term IVG culture of much smaller oocytes. IVG and xenotransplantation of small oocytes at a specific size will provide a new understanding of the mechanisms regulating oogenesis and folliculogenesis in the complex mammalian ovary.     

Effect of growth differentiation factor‐9 (GDF‐9) on the progression of buffalo follicles in vitrified–warmed ovarian tissues

To improve the reproductive performance of water buffalo to level can satisfy our needs, the mechanisms controlling ovarian follicular growth and development should be thoroughly investigated. Therefore, in this study, the expressions of growth differentiation factor‐9 (GDF‐9) in buffalo ovaries were examined by immunohistochemistry, and the effects of GDF‐9 treatment on follicle progression were investigated using a buffalo ovary organ culture system. Frozen–thawed buffalo ovarian follicles within slices of ovarian cortical tissue were cultured for 14 days in the presence or absence of GDF‐9. After culture, ovarian slices were fixed, sectioned and stained. The follicles were morphologically analysed and counted. Expression pattern of GDF‐9 was detected in oocytes from primordial follicles onwards, besides, also presented in granulosa cells. Moreover, GDF‐9 was detected in mural granulosa cells and theca cells of pre‐antral follicles. In antral follicles, cumulus cells and theca cells displayed positive expression of GDF‐9. In corpora lutea, GDF‐9 was expressed in both granulosa and theca lutein cells. After in vitro culture, there was no difference in the number of primordial follicles between cultured plus GDF‐9 and cultured control that indicated the GDF‐9 treatment has no effect on the primordial to primary follicle transition. GDF‐9 treatment caused a significant decrease in the number of primary and secondary follicles compared with controls accompanied with a significant increase in pre‐antral and antral follicles. These results suggest that a larger number of primary and secondary follicles were stimulated to progress to later developmental stages when treated with GDF‐9. Vitrification/warming of buffalo ovarian tissue had a little remarkable effect, in contrast to culturing for 14 days, on the expression of GDF‐9. In conclusion, treatment with GDF‐9 was found to promote progression of primary follicle that could provide an alternative approach to stimulate early follicle development and to improve therapies for the most common infertility problem in buffaloes (ovarian inactivity).     

In vitro Follicle Growth: Achievements in Mammalian Species

The exact mechanisms regulating in vivo folliculogenesis in mammalians have only been partly unravelled. Some processes, such as the initiation of growth of primordial follicles are still poorly understood. This increases the difficulty to culture follicles in vitro as the primordial follicles will be the ultimate starting material for culture.
There are important species differences in regulation and timing of maturation, which makes it difficult to transpose techniques.
Only in the mouse model, live pups were born when primordial or early preantral follicles were cultured entirely in vitro . Although no systems are as yet permitting complete in vitro culture of early follicle stages in large animals or humans, parts of folliculogenesis have been successfully reproduced in vitro . This review summarizes achievements of the last years in follicle culturing starting off at several stages of development.
Future applications of in vitro follicle culture include fertility preservation for humans, preservation of rare animal species and creation of oocyte banks for research.     

Regulation of secondary follicle growth by theca cells and insulin-like growth factor 1

Ovaries contain follicles at various stages of development, including primordial, primary, secondary, antral and Graafian follicles. Although the growth of these follicles is controlled to maintain regular ovulation, the mechanism through which this occurs remains unclear. In our study, we found that the growth rate of cultured secondary follicles separated from mice ovaries differed between follicles. After 4 days of culture, the size of some secondary follicles was markedly increased, while that of others had either slightly increased, remained unchanged or shrunk. We compared the expression levels of growth factors between these secondary follicles and found that the growth rate of cultured secondary follicles correlated with the expression level of insulin-like growth factor 1 (Igf1) mRNA. Igf1 mRNA expression level in secondary follicles containing theca cells was higher than that in secondary follicles without theca cells, and the granulosa cell proliferation around follicles containing theca cells was increased. Furthermore, an IGF1 inhibitor also inhibited the granulosa cell proliferation, and administration of IGF1 to secondary follicles without growth promoted granulosa cell proliferation. These results indicated that the theca cells of secondary follicles induced the expression of IGF1 and promoted the follicle growth.     

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。     

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。