甘氨酸对猪卵母细胞体外成熟质量及卵丘细胞的影响

本实验旨在研究甘氨酸对卵丘细胞及卵母细胞体外成熟质量的影响,优化猪卵母细胞体外成熟(IVM)培养体系。在猪卵母细胞体外成熟培养液中添加6 mmol/L甘氨酸,体外培养至44 h后,通过倒置显微镜测量卵丘细胞扩散直径,结合流式细胞术与qPCR检测卵丘细胞凋亡情况;进一步检测成熟卵母细胞中谷胱甘肽(GSH)、细胞成熟促进因子(MPF)和丝裂原活化蛋白激酶(MAPK)水平及细胞成熟相关基因表达。结果表明:甘氨酸能显著提高卵丘细胞扩散直径,减少卵丘细胞的总凋亡率,并能显著提高卵丘细胞中Bcl-2的mRNA表达;甘氨酸能显著增加成熟卵母细胞中的GSH、MPF和MAPK水平以及成熟相关基因BMP15、GDF9、CyclinB1、CDK1、C-MOS的mRNA表达。综上可知,甘氨酸可提高猪卵母细胞体外成熟质量,降低卵丘细胞凋亡率。     

卵丘细胞对猪卵母细胞成熟及发育的影响

本实验旨在研究有无卵丘细胞对卵母细胞成熟及其胚胎发育能力的影响。实验分别设裸卵组、卵丘细胞与卵母细胞共培养组、卵丘-卵母细胞复合体(COCs)组(对照组),检测猪成熟卵母细胞的存活率、极体率、孤雌激活后的卵裂率、囊胚率及各组卵母细胞的谷胱甘肽合成相关基因的表达。结果表明:COCs组卵母细胞经体外培养42 h后其存活率最高(95.76%),高于裸卵组(P<0.05)和共培养组(P>0.05);COCs组第一极体排出率为71.77%,高于共培养组和裸卵组(P<0.05);COCs组在后期发育中卵裂率与囊胚率高于共培养组和裸卵组(P<0.05),而共培养组的卵裂率及囊胚率高于裸卵组(P<0.05);与COCs组相比,裸卵组显著下调了GCLM、GCLC的表达(P<0.05),共培养组中GCLM的表达与COCs组无显著差异。研究结果显示,卵丘细胞对卵母细胞的成熟及发育均有显著影响,在卵母细胞成熟过程中,卵母细胞与卵丘细胞的缝隙连接受损会对卵母细胞的成熟效率产生影响,并且在卵母细胞后期发育中,缝隙连接的完整性与否会影响后期发育能力,并可推论出随着卵丘细胞的减少和缝隙连接的破坏,会造成谷胱甘肽合成基因表达降低,从而影响卵母细胞内的谷胱甘肽合成。     

牛卵丘细胞对卵母细胞体外成熟与孤雌发育的影响

试验以牛卵泡内卵丘-卵母细胞复合体(COCs)为材料,探讨了牛卵丘细胞包裹程度对卵母细胞体外成熟(IVM)和孤雌胚胎(PAEs)发育的影响。试验1,将COCs随机分为2组,在开展IVM前,将其中一组COCs的卵丘细胞机械吹打去除成为机械裸卵(DOs),研究卵丘细胞层存在与否对卵母细胞体外核成熟(排出第一极体)和孤雌激活后发育能力的影响;试验2,根据COCs卵丘细胞包裹层数将COCs分为3组,即卵丘细胞层少(1～4层)的COCs为A组、卵丘细胞层多(5层以上)的COCs为B组及包裹5层以上卵丘细胞且附带卵泡壁颗粒细胞层(FSP)的COCs为C组,研究卵丘细胞层包裹程度对卵母细胞的体外核成熟和孤雌激活后胚胎发育能力的影响。结果表明:卵丘细胞的存在更有利于卵母细胞体外成熟和孤雌胚胎发育;COCs中卵丘细胞包裹层数对卵母细胞体外核成熟率没有显著影响,但包裹卵丘细胞层数多且附带FSP的卵母细胞,其PAEs的囊胚率显著高于包裹卵丘细胞少的卵母细胞PAEs。     

猪卵丘卵母细胞复合体和壁层颗粒细胞上LHR/hCGR的检测及性腺激素的分泌

用取自 ３～５ ｍ ｍ 卵泡的处于减数分裂阻抑状态的猪卵丘卵母细胞复合体（ｐ Ｃ Ｅ Ｏ），培养 ４８ ｈ，用 Ｆ Ｓ Ｈ（１００ Ｉ Ｕ／ Ｌ）或 ｈ Ｃ Ｇ（０、５０、１００、２００、５００ Ｉ Ｕ／ Ｌ）刺激 ｐ Ｃ Ｅ Ｏ 分泌甾体激素；用放射受体测定法（ Ｒ Ｒ Ａ）比较 ｐ Ｃ Ｅ Ｏ 的卵丘细胞及壁层颗粒细胞（ Ｍ Ｇ Ｃ）上促黄体激素受体／人绒毛膜促性腺激素受体（ Ｌ Ｈ Ｒ／ｈ Ｃ Ｇ Ｒ）的数量； Ａ Ｍ ｅ Ｘ 石蜡切片、免疫组织化学染色，检测 Ｌ Ｈ Ｒ／ｈ Ｃ Ｇ Ｒ 在 ｐ Ｃ Ｅ Ｏ 以及 Ｍ Ｇ Ｃ 的分布情况。在 Ｆ Ｓ Ｈ 作用下，ｐ Ｃ Ｅ Ｏ 分泌的孕酮明显高于 ｈ Ｃ Ｇ 作用组和对照组 （ Ｐ ＜ ００５）； 平均每个壁层颗粒细胞上的 Ｌ Ｈ Ｒ／ｈ Ｃ Ｇ Ｒ是每个卵丘细胞的 １０５ 倍； Ｌ Ｈ Ｒ／ｈ Ｃ Ｇ Ｒ 在基膜两侧分布较多，且卵母细胞上没有 Ｌ Ｈ Ｒ／ｈ Ｃ Ｇ Ｒ，临近卵母细胞的卵丘细胞膜上较少被染色。以上结果表明，在体外试验中，可能由于 Ｃ Ｅ Ｏ 上 Ｌ Ｈ Ｒ／ｈ Ｃ Ｇ Ｒ 的数量不足或生理活性降低， Ｌ Ｈ／ｈ Ｃ Ｇ 不能促进卵母细胞恢复减数分裂。     

卵母细胞去核及卵丘细胞移核方法对猪体细胞核移植的影响

以猪体外成熟卵母细胞周围分离得到的卵丘细胞作为猪体细胞核移植的供核细胞,研究了卵母细胞不同去核方法(盲吸法、活性荧光染色去核法、盲吸法和活性荧光染色法联合去核法)的去核率差异,并比较了卵丘细胞透明带下注核和胞质内注核两种重建胚构建方法的胚胎裂解率、4 8细胞发育率和桑椹胚率的差异。结果表明:(1)以荧光染料染色法的去核率(87.18%)最高,与其他试验组相比差异显著(P<0.05);盲吸法去核所用时间最少(1.2 min/个),与其他试验组相比差异显著(P<0.05)。(2)透明带下注核比卵母细胞质内直接注核的卵裂率(40.11%/29.75%)和4 8细胞率(11.86%/6.81%)高,差异显著(P<0.05);桑椹胚率(1.75%/1.79%)无明显差异(P>0.05)。     

牛卵丘卵母细胞体外成熟的研究进展

牛体外受精技术是胚胎生物工程技术研究的基础,其中牛卵丘卵母细胞体外成熟培养是体外受精技术的关键,也是重要环节。自上世纪三十年代以来,国内外研究者们都付出了大量的研究工作,并取得了较大研究进展。目前,牛卵丘卵母细胞体外成熟培养体系已经确立,但其中的卵母细胞体外成熟和早期胚胎发育阻滞,卵母细胞的成熟调控机理等问题,还有待研究者们进一步深入探讨。本文主要从牛卵丘卵母细胞的获得方法、体外培养条件、体外培养液及添加物、存在问题等几方面来综述其研究进展。     

水牛卵丘颗粒细胞凋亡与卵母细胞体外发育潜能的研究

为了探讨水牛卵母细胞体外发育潜能与卵丘颗粒细胞凋亡之间的关系,本研究将水牛卵母细胞体外培养22～24 h后,取其卵丘颗粒细胞,采用Annexin V-FITC法进行颗粒细胞凋亡染色,进行凋亡率的分析,并与卵母细胞的成熟情况和卵母细胞的评分及形成卵裂、囊胚的结果进行对照.结果发现,随着卵母细胞成熟时间的增加,卵丘颗粒细胞凋亡率升高;随着卵母细胞评分和发育潜能的降低,卵丘颗粒细胞凋亡率逐渐升高.由此表明,卵母细胞发育潜能与卵丘颗粒细胞凋亡密切相关,通过测定卵丘颗粒细胞的凋亡率可预测未成熟卵母细胞的发育潜能.     

生长激素STH对体外培养猪COCs卵丘细胞影响的研究

试验研究了生长激素（somatotropin,STH）对猪卵丘-卵母细胞复合体（COCs）的卵丘细胞扩展、凋亡、凋亡相关基因Bcl-2、Bax表达的影响。结果表明,STH能够促进卵丘细胞扩展,抑制卵丘细胞凋亡,抑制凋亡相关基因Bax在卵丘细胞上的表达。     

Effect of Follicle Size on In Vitro Maturation of Pre‐Pubertal Porcine Cumulus Oocyte Complexes

Very small follicles (<3.0 mm diameter) are over‐represented on the surface of ovaries of non‐cycling pigs, and the oocytes collected from these follicles generally have reduced developmental competence in vitro. This study examined the effect of follicle size on the nuclear maturation (n = 608), the potential of parthenogenetic activation (n = 243) and the cyclic AMP (cAMP) content of pre‐pubertal porcine oocytes (n = 480). In addition, the influence of follicle size on steroid hormone synthesis was analysed. Cumulus oocyte complexes (COCs) flushed from small (2.5–4.0 mm) or large (4.5–6.0 mm) ovarian follicles were cultured for 0, 28 and 46 h. After 46 h of IVM, a greater proportion of oocytes from 4.5‐ to 6.0‐mm follicles reach metaphase II (MII) compared with those from follicles with 2.5–4.0 mm of diameter (96.1 vs 77.0%, respectively; p < 0.001). Parthenogenetic activation of oocytes from large follicles produced higher developmental rates than oocytes from large follicles (p < 0.05). At 28 h, the IVM medium with oocytes from large follicles contained significantly more 17ß‐oestradiol (E₂) than the medium with oocytes from small follicles (5.55 vs 3.45 ng/ml, respectively; p < 0.05) and at 46 h, the medium with oocytes from small follicles contained significantly more progesterone (P₄) than the medium with oocytes from large follicles (276.7 vs 108.2 ng/ml, respectively, p < 0.05). Porcine oocytes from large follicles have higher nuclear and cytoplasmic maturation capacities, but the differences did not appear to be cAMP‐mediated. Our findings also suggest that COCs from small follicles undergo more intensive luteinization than COCs from large follicles. The results show that oocytes from follicles with a diameter greater than 4.0 mm are more suitable for in vitro studies.     

Influence of the Cumulus and Gonadotropins on the Metabolic Profile of Porcine Cumulus–Oocyte Complexes During In Vitro Maturation

The aim of this work was to examine the influence of the cumulus and gonadotropins on the metabolic profile of porcine cumulus oocyte complexes (COCs) during in vitro maturation. Immature COCs were assigned to morphological classes A₁ (with a dense cumulus), A₂ (with a translucent cumulus), B₁ (with the corona radiata), B₂ (with only some remaining cumulus cells) and matured with or without gonadotropins. Glycolysis and ammonia production were higher in the A class COCs; gonadotropins increased both, especially in the A₁ COCs (p < 0.05). The A class COCs had the highest initial protein contents and at the end of in vitro maturation. Furthermore, hormonal stimulation induced a similar increase in protein contents of both A classes (p < 0.05). The neutral lipid content and reactive oxygen species (ROS) levels were similar in the immature oocytes of the COCs of all classes. A reduction was seen in both these variables when maturation proceeded either in the presence or absence of gonadotropins. The cumulus type surrounding the oocyte is related to the metabolism of carbohydrates and amino acids by the COC during in vitro maturation under gonadotropic stimulation. Oocyte lipolytic activity and ROS production appear to be independent of the surrounding cumulus and the presence of gonadotropins.     

水牛卵丘细胞和颗粒细胞单层对卵母细胞体外受精及其胚胎发育的影响

[目的]探讨卵丘细胞对水牛卵母细胞体外成熟、体外受精及颗粒细胞单层对水牛体外胚胎发育的影响。[方法]①按卵母细胞有无卵丘细胞情况分为五个组：即自然裸卵组（Ⅰ组）、自然裸卵＋卵丘细胞共成熟组（Ⅱ组）、人为裸卵＋卵丘细胞共成熟组（Ⅲ组）、AB级卵母细胞成熟后去除卵丘细胞受精组（Ⅳ组）、AB级卵母细胞对照组（Ⅴ组）分别进行体...     

猪卵母细胞冷冻保存效果的评判

建立快速、准确、有效的猪卵母细胞冷冻保存效果的判定方法,将有助于促进猪卵母细胞冷冻保存的研究,从而提高猪卵母细胞冷冻保存效果,为哺乳动物配子和胚胎工程方面的研究提供大量优质卵母细胞。作者主要介绍通过形态学观察结果和发育潜力来判定猪卵母细胞冷冻保存效果的一些方法,如检查冷冻解冻后猪卵母细胞形态和超微结构正常情况,或进行体外成熟、体外受精、孤雌激活、显微受精,然后据此对猪的卵母细胞冷冻保存效果进行评判。     

Effect of the Removal of Cumulus Cells on the Nuclear Maturation, Fertilization and Development of Porcine Oocytes

The present study was conducted to investigate the effects of attachment of cumulus cells to porcine oocytes during the process of maturation and fertilization on the nuclear maturation, fertilization and subsequent development after in vitro fertilization (IVF). In the first experiment, the cumulus cells were removed from cumulus-oocyte complexes (COCs) at 0, 24 and 42 h after the onset of maturation culture and were then cultured until reaching 42 h of cultivation. In the second experiment, COCs were denuded as described in the first experiment, then fertilized and cultured for 7 days. As a control, cumulus cells were allowed to maintain attachment to the oocytes until the end of IVF. The proportion of oocytes reaching metaphase II significantly increased with the delay in the removal treatment of cumulus cells. The proportion of normal fertilization gradually increased with delay in the removal treatment of cumulus cells from COCs until the end of IVF. However, no significant difference in the proportion of normal fertilization was found between the 42-h and control groups. The removal treatment of cumulus cells in the 0- and 24-h group significantly (p < 0.05) decreased the proportion of cleaved embryos when compared with the control, and none of them developed to the blastocyst stage. The proportion of development to the blastocyst stage was significantly higher (p < 0.05) in the control group than in the 42-h group (18.1% vs 12.4%; p < 0.05). The present study indicates that the attachment of cumulus cells to the oocyte during maturation and fertilization is important to support oocyte nuclear maturation, fertilization and subsequent embryo development. Particularly, the attachment of cumulus cells to the oocyte during IVF promotes embryonic development.     

卵丘细胞在卵母细胞体外成熟培养过程中的作用

介绍了卵丘细胞在卵母细胞体外成熟培养过程中的作用,主要从卵丘细胞与卵母细胞的结构与功能联系,卵丘扩展对卵母细胞体外成熟的影响及卵丘细胞层数对卵母细胞体外成熟的影响三个方面加以阐述。     

培养介质、卵丘细胞和卵泡直径对猪卵母细胞体外成熟的影响

A类卵母细胞在mTCM 199、NCSU2 3和NCSU37体系中培养 4 4～ 5 2小时后 ,成熟率分别为 76 .1%、78.1%和 6 5 .2 %。前两者差异不显著 (P >0 .0 5 ) ,但显著高于后者 (P <0 .0 5 )。卵母细胞在添加eCG和hCG的NCSU2 3体系中的成熟率 (75 .6 % )明显高于添加FSH的LH和成熟率 (6 5 .2 % ) (P <0 .0 5 )。A、B、C三类卵母细胞在NCSU2 3的成熟率分别为 73.3%、6 0 .4 %和 11.0 % ,三者间差异显著 (P <0 .0 5 )。大 (ф >6mm)、中 (ф =3～ 6mm)和小 (ф <3mm)三种卵泡中的卵母细胞在NCSU2 3中培养后 ,成熟率分别为 5 6 .2 % ,78.1%和 5 1.9% ,中等卵泡中卵母胞的体外成熟率显著高于其他两组 (P <0 .0 5 )。     

卵丘细胞对卵母细胞IVM及分子机制的研究进展

卵母细胞的成熟是当前胚胎工程及相关动物生物技术的基础及重要环节,对体外受精、转基因、胚胎克隆以及相关生物技术的发展具有重要的意义,本文主要将卵母细胞体外成熟的分子机制、卵丘细胞对卵母细胞成熟影响因素、目前取得成果以及存在的问题等方面进行归纳总结,为进一步的研究提供理论依据。