Modulation of Glycolysis and the Pentose Phosphate Pathway Influences Porcine Oocyte In Vitro Maturation

Glycolytic and pentose phosphate pathway (PPP) activities were modulated in porcine cumulus–oocyte complexes (COCs) during in vitro maturation (IVM) by the addition of inhibitors or stimulators of key enzymes of the pathways to elucidate their relative participation in oocyte maturation. The activities of glycolysis and PPP were evaluated by lactate production per COC and by the brilliant cresyl blue test, respectively. Glucose uptake per COC and the oocyte maturation rate were also evaluated. Lactate production, glucose uptake and the percentage of oocytes reaching metaphase II decreased in a dose‐dependent manner in the presence of the pharmacological (NaF) or the physiological (ATP) inhibitors of glycolysis (p < 0.05). The addition of the physiological stimulator of glycolysis (AMP) caused no effect on lactate production, glucose uptake or the meiotic maturation rate. The pharmacological (6‐AN) and the physiological (NADPH) inhibitors of PPP induced a dose‐dependent decrease in the percentage of oocytes with high PPP activity and in the nuclear maturation rate (p < 0.05). The physiological stimulator of PPP (NADP) caused no effect on the percentage of oocytes with high PPP activity. The glycolytic and PPP activities of porcine COCs and maturational competence of oocytes seem to be closely related events. This study shows for the first time the regulatory effect of ATP and NADPH as physiological inhibitors of glycolysis and PPP in porcine COCs, respectively. Besides, these pathways seem to reach their maximum activities in porcine COCs during IVM because no further increases were achieved by the presence of AMP or NADP.     

人源促卵泡素对猪卵母细胞体外成熟的影响

促卵泡素(FSH)一直在动物繁殖、体外培养及人类临床上广泛应用,但是无论是人源促卵泡素还是重组促卵泡素的效用、成分及活性都是不同的,由于这个原因,促卵泡素在实践中的应用一直没有一个规范的标准.试验着重研究了纯化于绝经后妇女尿液中的促卵泡素(hFSH)对猪卵母细胞体外成熟的影响,分别对猪体外成熟的卵母细胞进行地衣红染色和皮质颗粒的染色,来检测hFSH对卵母细胞的核及胞质成熟的影响,并检测了体外成熟猪卵丘细胞DNA凋亡的情况.     

马尾藻属藻类多糖对猪卵母细胞体外成熟及凋亡的影响

本研究就马尾藻属藻类多糖对猪卵母细胞的体外成熟及凋亡进行了探索:试验一证明,体外培养46h～48h时,成熟液添加马尾藻属藻类多糖能促进卵丘细胞扩展,添加20mg/ml马尾藻属藻类多糖能提高核成熟率(41.8%),与对照组29.3%的核成熟率相比,二者差异显著;试验二证明,添加马尾藻属藻类多糖可延长猪卵母细胞体外存活时间,体外培养第16d,在含有0、5mg/ml、20mg/ml马尾藻属藻类多糖培养液中的猪卵母细胞存活率分别为2.6%、18.9%、23.0%,添加与否差异显著。     

马尾藻类多糖对猪卵母细胞体外成熟及孤雌激活的影响

就马尾藻类多糖对猪卵母细胞体外成熟及孤雌激活的影响进行了探索。试验1证明。体外培养时。成熟液添加马尾藻类多糖能促进卵丘细胞扩展，成熟培养47-48h，添加20g／L马尾藻类多糖能提高核成熟率(41．8％)，与对照组29.3％的核成熟率相比，二者差异显著；试验2证明，进行化学联合激活时，添加20g／L马尾藻类多糖(未添加LH)W获得60.4％孤雌激活分裂率。显著高于对照组42．6％的孤雌激活分裂率。     

猪卵母细胞体外成熟培养的主要影响因素

猪卵母细胞的体外成熟是猪体外受精技术的基础环节，随着克隆、转基因等高新生物技术的发展，尤其是猪在人类医学研究和临床中潜在的应用前景，已使该技术成为近年来的研究热点。本文主要就卵母细胞成熟培养的影响因素作一综述，供参考。     

卵母细胞成熟及其成熟调控因子

卵母细胞成熟过程是个复杂的减数分裂过程,许多因子参与这个过程的调控.为了探讨哺乳动物卵母细胞体外成熟的机理,指导家畜体外胚胎生产,本文就卵母细胞体外成熟培养(in vitro maturation IVM)及影响因子等方面进行综述.认为在卵母细胞体外成熟过程中,仍需对胞质成熟、基因调控及影响因子等方面进行深入研究.     

猪卵母细胞体外成熟和孤雌激活参数研究

本研究比较了按不同方法贮藏的培养液对猪卵母细胞体外成熟及成熟后孤雌激活对胚胎发育的影响。此外,还探索了针对初情期前母猪体外成熟卵母细胞和孤雌激活方案。结果如下：①1.4 kv/cm、100 μs、1DC电激活后,卵母细胞死亡率明显高于2.0 kv/cm、30 μs、1DC和2.0 kv/cm、60 μs、1DC处理组,但激活后胚胎卵裂率和囊胚率相似;②使用钙离子载体和6-二甲基氨基嘌呤（Ionomycin+6-DMAP）处理后,胚胎卵裂及囊胚率都明显不如电激活处理;③6次独立试验结果证明：经4℃冷藏和-20℃冷冻保存的培养液用于猪卵母细胞培养,其体外成熟率、孤雌激活后的卵裂率及囊胚发育率无显著差异（P＞0.05）。说明成熟卵在2.0 kv/cm、30 μs、1DC或者 2.0 kv/cm、60 μs、1DC电击参数激活下可以降低死卵率;按本试验设计的电激活方案处理初情期前母猪成熟卵优于化学激活;在-20℃冷冻保存猪卵母细胞成熟液是可行的。      

不同培养时间对猪卵母细胞体外成熟效果的影响

试验回收中等卵泡(2～6mm)中的A、B级卵母细胞,然后把卵母细胞分为12组移入培养液TCM-199进行体外成熟培养。A组在不同培养时间(30,36,42,45,48,51h)和B组在不同培养时间(32,38,44,47,50,53h)用H33342染色,倒置显微镜下观察卵母细胞核成熟情况;再经电激活处理,观察卵裂率。结果表明:45h和48h的A级卵母细胞成熟培养后的成熟率差异不显著(P>0.05);47h和50h的B级卵母细胞成熟培养后的成熟率差异不显著(P>0.05);48h的A级卵母细胞和50h的B级卵母细胞卵裂率差异不显著(P>0.05)。     

活性氧对胚胎体外发育阻滞的影响

自由基是含有一个不成对电子的原子团.早在1931年,Michalis就提出某些酶促氧化还原反应的中间产物为自由基,其试验证据也表明,生物体内可能存在自由基,然而直到自由基生物学迅猛发展的最近30年,自由基在机体内的产生与清除才得以重视.现在,人们已经认识到自由基与许多疾病的发生和发展密切相关.自由基主要分为活性氧自由基和活性氮自由基.文章主要介绍活性氧自由基.     

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.     

换液培养对猪卵母细胞的成熟及胚胎体外发育的影响

通过后期去除激素培养比较了卵丘卵母细胞成熟及构建克隆胚后的发育情况,同时孤雌胚胎和体外受精胚胎体外培养48 h后全量换液,比较了其卵裂率和囊胚率之间的差异。结果显示,卵丘卵母细胞培养22h后换成不含激素的成熟培养液继续培养至44 h,其成熟率与对照组无显著差异(P0.05);后期克隆胚胎卵裂率和囊胚率与对照组均无显著差异(P0.05)。孤雌胚胎换液培养卵裂率高于未换液组,囊胚率低于未换液组,但差异均不显著(P0.05)。体外受精胚胎换液培养卵裂率以及囊胚率和未换液组差异不显著(P0.05)。本实验结果说明换液培养对卵母细胞的成熟及后续的体细胞克隆胚发育无显著影响,孤雌和体外受精胚胎换液培养对后期发育也无显著影响。     

白牦牛卵母细胞体外成熟的研究

研究了不同激素配比、性周期阶段对白牦牛卵母细胞体外成熟的影响，同时研究了不同培养基对早期胚胎体外发育的影响。结果表明：M199＋0．2mmol／L丙酮酸钠＋10％NBs＋5．0mg／L LH＋0．5mg／LFSH＋1＞g／ml 17β-E2是白牦牛卵母细胞体外成熟较为理想的培养系统（成熟率79．4％、卵裂率42．5％）。采集白牦牛卵母细胞时卵巢所处性周期阶段（卵泡期、黄体期）影响卵母细胞的体外成熟（成熟率分别为77．6％、69．5％，卵裂率分别为45．7％、35．6％）。输卵管上皮细胞和颗粒细胞是理想的共培养细胞，能有效克服白牦牛早期胚胎发育阻滞（桑囊胚发育率分别为10．6％、7．7％）。     

猪卵母细胞体外成熟过程中p90rsk未被MAPK激活

从屠宰场获得的初情期母猪卵巢中的卵母细胞 ,在修改后的 TCM199中进行体外成熟培养。在卵母细胞发育的一定时期取样 ,利用 SDS- PAGE蛋白质电泳和免疫印迹技术 ,研究了 MAPK(丝裂原激活蛋白激酶 ,又称 ERK)和p90 rsk (ribosomal S6 kinase)对猪卵母细胞成熟的调节。结果表明 ,修改后的 TCM199可以满足猪卵母细胞成熟的需要 ,但猪卵母细胞在体外成熟过程中 ,p90 rsk未随 MAPK激活而激活 ,说明维持猪卵母细胞的 M 阻滞与 MAPK有关 ,而与 p90 rsk无关。     

牛卵母细胞体外成熟培养

本文着重探讨了牛卵母细胞的体外成熟培养，对卵子的发生、卵母细胞体外成熟的原理进行了阐述，叙述了从卵母细胞的回收、选择到成熟等步骤。本实验采用磷酸缓冲液(PBS)冲洗卵巢、吸卵，以含有FSH、LH、雌二醇等成分的成熟液对卵母细胞进行体外成熟培养，使卵丘细胞扩散，第一极体排出，卵母细胞达到成熟。     

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。     

Effect of Lycopene on Cytoplasmic Maturation of Porcine Oocytes In Vitro

The aim of the present study was to improve cytoplasmic maturation of porcine oocytes by the addition of lycopene into in vitro maturation (IVM) media. We designed six experimental groups; IVM medium was supplemented with 10 IU/ml FSH, FSH and 10 IU/ml human chorionic gonadotrophin (hCG), or FSH and 7 μm lycopene in the first half of the IVM culture (0–22 h) followed by further culture (22–44 h) with or without hCG. The addition of lycopene into IVM media delayed the interruption of communication between an oocyte and the cumulus cells. Although meiotic competence was similar among the six groups, the glutathione level of matured oocytes was significantly higher in the lycopene‐supplemented group (9.89 pmol per oocyte) than that in other groups (7.25 and 7.81 pmol per oocyte). Fertilization rate was significantly improved in lycopene‐supplemented groups (58.3%) more than that in the group supplemented with FSH only (43.1%), whereas there were no differences in developmental competence among the groups (blastocyst rate: 20.1–29.5%). These results indicate that insufficient cytoplasmic maturation during conventional IVM resulted by disconnection of the gap junction between an oocyte and the cumulus cells in the early phase during IVM culture. We concluded that lycopene induced a prolonged sustainment of gap junctional communication between an oocyte and the cumulus cells during porcine IVM culture, which was an effective cytoplasmic maturation of porcine IVM oocytes.     

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.     

影响猪卵母细胞体外成熟的因素

就影响猪卵母细胞体外成熟的因素, 如卵巢获取、卵母细胞来源、卵丘细胞、培养时间、培养条件基础培养基、激素、血清和卵泡液等, 及其研究进展进行了综述。     

间隙连接对牛卵母细胞体外成熟的影响

该试验通过对牛卵母细胞不同形式的处理,讨论细胞间隙连接对于其体外成熟的影响作用.在最适于卵母细胞体外成熟的浓度(1.2 IU/mL)条件下,发现卵母--卵丘细胞之间的间隙连接对卵母细胞的成熟有着直接的影响,并且是体外成熟的一个有利条件.