卵丘

During denuded mice oocyte mature process in vitro, the cortical granulesmay have abnormal numbers or generation problems. This may be another reason thatmakes denude oocytes matured in vitro have a clearly deficiency on fertilization invitro.

3去除卵丘细胞的小鼠卵母细胞在体外成熟过程中，皮质颗粒可能存在继发性生成障碍或者数量异常，该结果可能是去除卵丘细胞的小鼠卵母细胞存在体外受精障碍的原因之一。

The results shown as follow: 1. The study on mice denuded oocytesin vitro maturation, in vitro fertilizationand in vitro development. This part of paper is to discuss whether the remove of granulose cells can affect the mice oocytes maturation rates, fertilization rates and development rates in vitro.

一、去除卵丘细胞的小鼠卵母细胞的体外成熟、体外受精初步研究本实验探讨了不同成分明确的成熟培养液对去除卵丘细胞的小鼠卵母细胞的体外成熟、体外受精和胚胎的体外发育的影响。

Partial or complete removal of cumulus before fertilization had no effect on fertilization rates of oocytes, but complete removal of cumulus impaired embryo development, leading to reduced morula/blastula rates.

受精前去掉山羊卵丘或者去掉部分卵丘对受精率没有显著影响，但是受精前完全去掉卵丘对于胚胎的后续发育有明显影响。

Hormone level in maturation medium affected cumulus expansion of COC, but cumulus expansion became less dependent on hormone level with time of culture. Porcine immatured oocytes, granulosa cells from small antral follicles and cumulus cells of immatured oocytes secreted cumulus-expansion enabling factors. Granulosa cells from 3-6mm follicles did not produce CEEF, but there was some CEEF in the follicular fluid of this sized follicles.

激素水平对卵丘扩展有影响，但随培养时间的延长，对激素的要求有逐步减少的趋势；猪卵丘扩展不依赖于卵母细胞，卵母细胞核成熟也与卵丘扩展无关；GV期卵母细胞、小腔卵泡内颗粒细胞和成熟卵的卵丘细胞都有较高的分泌CEEF能力；3-6mm卵泡的壁颗粒细胞不能分泌CEEF，但3—6mm卵泡的卵泡液中有CEEF。

Hormone level in maturation medium affected cumulus expansion of COC,but cumulusexpansion became less dependent on hormone level with time of culture.Porcine immaturedoocytes,granulosa cells from small antral follicles and cumulus cells of immatured oocytessecreted cumulus-expansion enabling factors.Granulosa cells from 3-6mm follicles did notproduce CEEF,but there was some CEEF in the follicular fluid of this sized follicles.Sun xingshen, Animal Histology and Embryology

激素水平对卵丘扩展有影响，但随培养时间的延长，对激素的要求有逐步减少的趋势；猪卵丘扩展不依赖于卵母细胞，卵母细胞核成熟也与卵丘扩展无关；GV期卵母细胞、小腔卵泡内颗粒细胞和成熟卵的卵丘细胞都有较高的分泌CEEF能力；3-6mm卵泡的壁颗粒细胞不能分泌CEEF，但3-6mm卵泡的卵泡液中有CEEF。

Under the transmission election microscope, we had observed that the state of oocytes arid the cumulus cells around them was tightness, and there were plenty of long and thin microvilli playing the role of connection in the enwrapping. The shape of cumulus cells nucleus was ovate, and the longer and shorter diameters of the cumulus cells were 4.250±0.042μm and 2.750±0.07 μm respectively. The zona pellucida was well proportioned and tightly linking with oocyte. There were great deals of multi-crista mitochondria, lipid droplet and vacuolus. The mitochondria longer and shorter diameters were 0.600±0.106μm and 0.490±0.117μm. Granular nucleoplasma distributed very well in the cell nucleus.

电镜下，卵母细胞外的卵丘细胞紧紧包裹卵母细胞，且与卵母细胞之间有大量细长的微绒毛相联系；卵丘细胞核呈卵圆形，其长径为4250±0.042μm，短径为2.750±0.071μm；透明带均匀，与卵母细胞结合紧密；在皮质区集中分布大量的多峰型线粗体、脂滴和空泡，线拉体长径为0.600±0.106μm，短径为0.490±0.117μm；细胞核中颗粒状的核质分布非常均匀。

Our study includes four aspects. In the first aspect we study several important conditions of porcine oocytes maturation in vitro and oocytes cleavage after parthenogenetic activation and found mNCSU-23+15IU/mlPMSG+20IU/mlHCG+15% PFF+0.57mMcysteine is a good culture condition .When the Cocs are cultured in it ,the maturation rate and oocytes cleavage rate are higher than those of foreign covered. Our result are (86.7±3.35)% and (86.3±4.16)% and the highest report of foreign is(85.7±4.1)%.In the second aspect we study the effect of different chemical activations on development of porcine parthennogenetic embryo and found two best activation method. The first one is that putting the maturation MII oocytes in the 20μmol/L ionomycin for 30 minutes and then putting them in the NCSU-23 condition containing 5μg/mICB and 5mM/L6-DMAP for 3.5 hours, the oocytes cleavage rate and morulae/blastocysts development rate are (76.7±7.6)% and (37.1±6.4)%.The second one is that putting the maturation MII oocytes in the 200μM/L Thimerosal for 20 minutes and then putting them in the NCSU-23 condition containing 8mM DTT for 30 minutes

本研究分为4个部分，第一部分对影响猪卵母细胞体外成熟和孤雌激活后胚胎分裂的几个重要条件进行了比较研究，确立了一种较好的培养方法：与颗粒细胞共培养，找到了一种适合猪卵母细胞体外成熟的培养基：mNCSU-23+15IU/mlPMSG+20IU/mlHCG+15%PFF+0.57mM半胱氨酸，成熟率和分裂率分别为(86.7±3.35)%和(86.3±4.16)%，国外报道的最高成熟率为(85.7±4.1)%；第二部分对猪卵母细胞孤雌激活的化学方法进行了研究，确立了化学激活猪卵母细胞的两种最佳方法：1将成熟的去卵丘颗粒细胞的MII期卵母细胞用20μmol/Lionomycin作用30min，再将卵母细胞培养于含5μg/mlCB和5mM/L 6-DMAP(6-二甲基氨基嘌呤)的NCSU-23培养液中，卵裂率和桑囊胚发育率达到(76.7±7.6)%和(37.1±6.4)%2将成熟的去卵丘颗粒细胞的MII期卵母细胞在200μM/L的Thimerosal中处理20min，再与8mM的DTT共孵育30min，卵裂率和桑/囊胚形成率为(81.0±2.8)%和(39.6±2.7)%；第三部分对孤雌激活胚胎的培养条件进行了研究，确立了一种最佳的胚胎培养条件：在SOF简单培养基中添加颗粒细胞进行前3天的培养，然后转入添加胎牛血清的NCSU—23培养基并和输卵管上皮细胞进行后期的培养，其桑椹胚和囊胚的发育率为(59.5±3.2)%；第四部分研究了IGF-I

In the present study, we collected cumulus cells oocyte complex from ovaries of two different strain mice. The cumulusenclosed oocytes were cultured for 6 h in MEM supplemented with growth factor and FSH. The meiotic maturation of these oocytes has progressed to pro-metaphse Ⅰ stage and the condensed chromosomes are visible under DIC microscope, metaphase Ⅰ spindle even can be detected under Polscope. The metaphase Ⅰ spindles of oocytes were exchanged under such microscopes. After electric stimuli, 91. 6% and 91. 6% karyoplasts-cytoplasm pairs were fused respectively. The resulting oocytes were cultured further in MEM and over 80% of oocytes released the first polar body. 79% and 77% of oocytes formed two pronuclei after in vitro fertilization and the embryos were cultured in KSOM supplemented with amino acids. Over 60% of embryos developed to blastocyst stage.

在本研究中我们在取得两种不同品系小鼠的卵丘卵母细胞复合体后，先将卵丘卵母细胞复合体置于含有多种生长因子和激素的MEM培养液中培养6小时，此时卵母细胞已进入第一次减数分裂的前中期，并且在DIC倒置显微镜下可以看到浓缩的染色体，用Polscope可以发现明显的纺锤体，借助这种显微镜通过显微操作将两种不同品系小鼠来源的卵母细胞的MI纺锤体进行互换，经过三次直流电脉冲作用后，分别有91.6％的胞质—MI核质体对融合，经过进一步的培养后，超过80％的重组卵母细胞排出第一极体，体外受精后分别有79％和77％的重组卵形成双原核，受精后的胚胎在KSOM胚胎培养液中体外培养4天后，超过60％的胚胎发育至囊胚。

The results show that: 1 supplementation of protein to maturation media improves cumulus expansion in vitro compared to the protein-free control, but cumulus expansion is not necessarily related to oocyte nuclear maturation in pigs, and cumulus expansion is not the criterae for determination of nuclear maturation of pig oocytes, but only the exclusion of the first polar body; 2 exposure of pig COCs to hormone supplements for 23-24 hours improved cumulus expansion but had no significant effect on nuclear maturation compared to that for 46-48 hours; 3 under our research conditions, supplementation of different proteins into different maturation media has different effects on porcine oocyte nuclear maturation, but has no significant effect on subsequent embryonic development after IVF; 4 the nuclear maturation rates of pig oocytes matured in mTCM+pFF and mNCSU+pFF are superior than that in mNCSU+FCS; 5 different maturation media have no effect on pig oocyte cumulus expansion and subsequent embryonic development after IVF.

结果显示：(1)在成熟液中添加蛋白质可以加强卵丘细胞的扩散，但猪卵母细胞的核成熟与其周围的卵丘细胞扩展没有必然的联系，卵丘细胞扩散或成放射状不宜作为猪卵母细胞核成熟的标准，只有排出第一极体才能作为猪卵母细胞核成熟的标志；(2)在猪COCs的46-48小时成熟培养的后23-24小时阶段去除成熟液中的激素不但可以保证卵母细胞的核成熟率，而且可加强卵丘细胞的扩散；(3)在现有实验条件下，在mTCM和mNCSU中添加10％pFF与在mNCSU中添加10％FCS相比可获得较高的猪卵母细胞核成熟率；(4)在不同的成熟液中添加不同的蛋白质对猪卵母细胞核成熟率的影响效果不一样，但对体外受精后的早期胚胎发育影响不明显；(5)成熟液种类对猪卵母细胞的卵丘细胞扩散和体外受精后的早期胚胎发育无显著影响。

The cumulus cells of OCC were cut off and dispersed by 1 mL syringe. The cumulus cells were co-cultured with the immature oocytes retrieved from the COH cycles after they adherent to the bottom of the dish. The immature oocytes were experienced IVM procedures in different culture media. They were divided into 3 groups(the oocytes at germinal vesicle stage from one woman were allotted to the same group randomly). Group 1: basic culture medium+ human follicular fluid; Group 2: solution A+ cumulus cells; Group 3: solution A+ OCC+ follicle stimulating hormone + epidermal growth factor. Then, the maturation rate, fertilization rate and formation rate of available embryo were observed.

在控制性促排卵周期有未成熟卵母细胞时，将同周期成熟卵丘复合体切出部分卵丘细胞，用1 mL注射器抽打分散细胞，贴壁培养。113个治疗周期中，298枚生发泡期卵母细胞经3种不同培养液体外成熟培养(同一病人的生发泡期卵被随机分到某同一组中)：第1组28个周期中73枚：基础培养液+卵泡液；第2组40个周期中115枚：A液+分散贴壁的卵丘细胞；第3组45个周期中110枚：A液+分散贴壁的卵丘细胞+促卵泡生成激素+表皮生长因子。

In the United States, chronic alcoholism and hepatitis C are the most common ones.

在美国，慢性酒精中毒，肝炎是最常见的。

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如果有任何问题，你可以随时联系我。