卵母细胞

Oogonium develops into early oocyte in the ovary, and then the oocyte leaves the ovary for the coelomic fluid in the form of single cell or cell mass followed by the rapid separation of the group of oocytes into individual ones. Oocyte enters into the nephridium after its maturation. The rupture of germinal vesicle marks the oocyte maturation. Oocyte in the coelom does not have follicle membrane and vitelline membrane is formed and developed by the oocyte itself. Smaller oocyte (0μm in diameter) is round, and larger ones (≥60μm in diameter) is ovate. The short and long diameters of a morphologically mature oocyte are about 115—120μm and 140—145μm respectively, and the vitelline membrane is 7—9μm thick.

卵原细胞在卵巢中发育至早期卵母细胞时期单个或成团脱离卵巢入体腔液中，卵母细胞团细胞很快分离为单个细胞；卵母细胞在体腔液中发育成熟后进入肾管；生发泡破裂是卵母细胞成熟的标志；体腔中卵母细胞无滤泡膜，卵黄膜的形成与发育靠卵母细胞本身；卵径小于60μm的卵母细胞呈圆形，卵径大于60μm 的卵母细胞为卵圆形，形态上成熟的卵母细胞短径约115—120μm、长径约140—145μm、卵黄膜厚7—9μm。

The oogenesis of P. esculenta can be divided into the following phases in morphological characteristics of oogonium and oocyte development: the proliferative phase of oogonium (0μm in diameter), the initial growth phase of oocyte (10—20μm in diameter), the grand growth phase-Ⅰof oocyte (20—60μm in diameter), the grand growth phase-II of oocyte (60μm×70μm to 120μm×145μm in size), the mature phase of oocyte, and the declining phase, showing a dynamic changes in oogenesis of P. esculenta .

以卵原细胞与卵母细胞发育的形态学特征为依据，将可口革囊星虫的卵子发生过程划分为：卵原细胞增殖期(卵径0μm)、卵母细胞小生长期(卵径10—20μm)、卵母细胞大生长期Ⅰ(卵径20—60μm)、卵母细胞大生长期Ⅱ[大小为(60μm×70μm)—(120μm×145μm)]、卵母细胞成熟期及退化期6 个阶段，反映了可口革囊星虫卵子发生过程的动态变化。

Different strains have different reaction to the delay of oviducts in body after animal death. After 10min delay C57BL/6 mouse oocytes have death rate of 56.5%,significantely higher than Kunming mouse (47.6%);Spontaneous activation rates were respectively 13.3% and 46.0%, C57BL/6 were obviously lower than Kunming mouse.4. The oviducts were obtained after being delayed 5min 24h after the mice were injected with hCG. The oocytes were cultured in CZB. About 81.1% occurred spontaneous activation, evidently lower than parthenogenetic rate (96.4%) with SrCl_2. Spontaneous activable oocytes had high cleavage rate(93.2%) and 4-cell rate(87.3%). However, spontaneous activable oocytes had blastula development rate(18.7%) as low as parthenogenetic oocytes by SrCl_2(22.9%).

不同品系小鼠卵母细胞对输卵管在体内滞留产生的反应不同，滞留10min C57BL/6系小鼠卵母细胞死亡率为56.5%，显著高于昆明鼠(47.6%)；自发激活率分别为13.3%和46.0%，C57BL/6系小鼠显著低于昆明鼠。4.hCG后24h体内滞留5min卵母细胞在CZB中培养自发激活率为81.1%，显著低于SrCl_2孤雌激活率(96.4%)；自发激活的卵母细胞有较高的卵裂率(93.2%)和4-cell比率(87.3%)，但囊胚率(18.7%)较低，同卵龄的卵母细胞经SrCl_2孤雌激活囊胚发育率为22.9%，差异不显著。

This experiment aimed to systematically investigate and analyze the ethanol activated rates of mouse oocytes, affected factors of oocytes activation, types and development of parthenotes, and the organization and roles of the cytoskeleton during pronuclear formation and migration and early embryo cleavage by ethanol activation of oocytes, embryo culture in vitro and immunofluorescence cyto-chemistry. The results:(1) The ethanol activation of mouse oocytes showed that the activated and developmental rates of oocytes increased significantly with the increase of ethanol concentration and extension of exposure time, but over concentration and exposure time would result in increased fragment rates significantly. 7% ethanol treated oocytes for 7min was the optimum activated condition.

本实验主要利用卵母细胞的乙醇激活、胚胎体外培养和免疫荧光细胞化学方法对小鼠卵母细胞乙醇激活效率、影响因素、孤雌胚类型、发育、原核形成、迁移及早期胚胎卵裂过程中细胞骨架的组装、作用等进行了系统的研究和分析，结果显示：(1)小鼠卵母细胞的乙醇激活结果表明，随着乙醇浓度的升高和作用时间的延长，卵母细胞激活率和发育率都显著提高，但乙醇浓度过高和作用时间过长会导致卵母细胞碎裂率的显著增加，7%乙醇作用卵母细胞7min 为最佳激活条件。

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

Following chemical activation, blastocysts rate of the treated oocytes was similar to untreated oocytes.8 Following fertilization, however, few oocytes inhibited with CHX developed into morulae/blastocysts, due to a high incidence of polyspermy.9 Cortical granule migration occurred during inhibition, but CHX inhibition impaired CG migration, significantly no oocytes inhibited by CHX completed CG migration after maturation.10 CHX inhibition had no effects onα-microtubles and microfilaments of goat oocytes.

抑制24h转为正常培养24h，不影响卵母细胞的成熟和孤雌激活能力，并且CHX抑制后再成熟的卵母细胞经孤雌激活发育到囊胚的比例与对照组卵母细胞相似。8、体外受精后，CHX抑制后再成熟卵母细胞的多精受精现象显著增加，发育到桑椹胚/囊胚的比例显著低于对照组。9、CHX抑制过程中皮质颗粒仍能发生迁移，但是CHX抑制会对皮质颗粒的迁移造成不可恢复的损伤，使再成熟的卵母细胞内皮质颗粒不能完全迁移。10、CHX抑制对山羊卵母细胞α-微管和微丝都没有影响，无论是抑制后处于生发泡期的卵母细胞，还是抑制后再成熟的卵母细胞，微管和微丝的分布都与对照卵母细胞相似。

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。

Pig oocytes cultured in vitro for some time were inseminated by frozen–thawed ejaculated sperm. At specified times after insemination, sperm penetration, cell cycle progression and mitogen-activated protein kinase phosphorylation were evaluated. It was shown that:(1) oocytes at various maturational stages could be penetrated by sperm;(2) sperm penetration did not affect meiotic cell cycle progression;(3) sperm penetration of germinal vesicle oocytes and maturing oocytes did not alter MAPK phosphorylation; and (4) when premetaphase I and metaphase I oocytes, in which MAPK was activated, were fertilised, no evident MAPK dephosphorylation was detected as in metaphase II oocytes.

不同成熟阶段的猪卵与精子融合后体外培养发现：(1)不同成熟阶段的猪卵母细胞都可被精子穿透；(2)精子穿透不影响减数分裂细胞周期进程；(3)精子穿透GV期卵母细胞和正在成熟的卵母细胞不改变MAPK磷酸化：(4)已受精的前中期I和中期I卵母细胞的MAPK有活性，MAPK不像MII卵母细胞那样受精后发生磷酸化。

Japonicus.Nerve extraction substance of sea star and dithiothreitol has been reported to be effectively to induce oocyte maturation in sea cucumbers. In this study we tested their effects on oocyte maturation induction in sea cucumber A. japonicus. The results are as follows, oocyte of A. japonicus does not get mature spontaneously. Neither NES alone nor NES with follicle suspension can induce oocyte maturation. While DTT can prominently increase the percentages of germinal vesicle break down. When the concentration of DTT is between10-1 mol/L and 10-3 mol/L, the effect of maturation induction is more sufficient. When the concentration is about 10-2 mol/L, the percentage of GVBD can reach up to more than 90%.

分别利用在海参中有诱导作用的海星神经提取液和二硫苏糖醇对从成熟卵巢解剖出的刺参卵母细胞进行诱导成熟试验，结果显示，刺参的卵母细胞在海水中不会发生自发成熟，海星神经提取液以及神经提取液加滤泡悬浮液对卵母细胞没有诱导成熟的作用，而二硫苏糖醇处理可以诱导卵母细胞生发泡破裂，当DTT溶液的浓度处于10-1 mol/L到10-3 mol/L之间时，对刺参卵母细胞的促熟作用较为明显，在10-2 mol/L左右时，卵母细胞的诱导成熟率可以达到90%以上。

Do you know, i need you to come back

你知道吗，我需要你回来

Yang yinshu、Wang xiangsheng、Li decang,The first discovery of haemaphysalis conicinna.

1〕 杨银书，王祥生，李德昌。安徽省首次发现嗜群血蜱。