作者要感谢Fabrice Vincent对激光扫描共焦显微镜（LSCM）的支持，PhilippeBarrière和Thierry Blard进行每日超声卵巢扫描和hCG注射。这项工作部分得到"区域经济和区域伦巴第"项目"Ex Ovo Omnia"（AML授权号26096200）的支持。奖学金（2012年至2012年合约），FP7-PEOPLE-2011 CIG，研究执行机构（REA）"Pro-Ovum"（授予VL的第303640号）;"欧莱雅意大利"以及由欧洲社会基金，2007 - 2013年人力资源开发部门行动计划（合同编号:POSDRU / 89 / 1.5 / S / 62371至IM）共同资助的农业和兽医学博士后。 体内卵母细胞收集由Institut du Cheval et de l&#39;Equitation提供资金。

所有程序均经动物保护使用委员会CEEA Val de Loire No.19批准，并按照"实验动物护理和使用指导原则"进行。 卵母细胞收集和体外成熟<ol><li> 卵子接送 <ol><li>每日通过超声检查成年母体队列中卵巢卵泡的直径。在卵泡≥33mm（优势卵泡）出现时，在颈静脉上部注射1500IU人绒毛膜促性腺激素（hCG）的母马（iv）。 <ol><li>在进行注射前，用酒精拭拭该区域，找到颈静脉沟，并将拇指置于注射部位2-3厘米处，以引起静脉上升。将针几乎平行于颈部插入并抽出，以确保针在静脉中（血液将进入注射器）。此时，注射; hCG会诱导m优势卵泡卵母细胞的成熟。 </li></ol></li><li>注射hCG后35 h，用detomidine（15μg/ kg，iv），酒石酸丁醇（10μg/ kg，iv）和丁基东莨菪碱溴（0.2-0.3 mg / kg，15 mL / mare，iv）镇静母体。 </li><li>将针连接到超声波探头。将超声波探头插入阴道，并将卵巢经直肠对准超声探头。指示一名操作人员操纵针头，另一名操作者将卵巢固定在适当位置，使卵泡面向超声波探头。从具有优势卵泡的卵巢开始。 </li><li>用针穿刺阴道壁和优势卵泡壁（OPU的超声波探头配备有连接到抽吸系统的针的通道）;然后在回波图像中可见。 </li><li>将滤泡液吸入与抽吸系统连接的50mL锥形管中。倒入圆锥形内容物管成大型培养皿，并使用立体显微镜搜索卵丘卵母细胞复合物（COC）。 </li><li>由于COC并不总是与滤泡液一起回收，所以用含有5IU / mL肝素37℃的Dulbecco改性磷酸盐缓冲盐水（DPBS）冲洗滤泡。检查DPBS的COC存在，如先前在步骤1.1.5中针对滤泡液所述。冲洗数次直到检索到COC。 </li><li>一旦来自优势卵泡的COC被取回，穿刺并冲洗≥5和≤25mm的卵泡，如步骤1.1.3-1.1.4中对优势卵泡所述;卵泡≥5和≤25不表达促黄体激素/绒毛膜促性腺激素受体（LHCGR），因此，它们的卵母细胞不会响应于hCG而成熟，并且将在GV阶段（未成熟）收集。只保留COC与几层完整的卵丘细胞和棕色，细粒状的卵子。舍弃其他人</li><li>在OPU的最后，在用苄青霉素15000IU /动物注射母体以预防感染。 </li><li>将母马安置在一个安静，干净的稳定下至少2小时。通过确定耳朵的位置，对刺激的反应（ 例如噪音）和步态，在将母马返回其他动物的公司之前检查意识的迹象。 </li></ol></li><li> 体外成熟 <ol><li>补充有1,790IU / L肝素，0.4％牛血清白蛋白（BSA），青霉素和链霉素的HEPES缓冲TCM199（20mM Hepes）至37℃。提前准备这种介质。过滤消毒，并保持在4°C长达6个月。 </li><li>通过补充含有50ng / mL表皮生长因子（EGF）和20％小牛血清7的 NaHCO 3缓冲的TCM199（25mM NaHCO 3 ）来制备IVM培养基。打开新瓶后3周内使用NaHCO 3缓冲液TCM199。预先准备EGF作为100x股票，冻结在-20°C，并在6个月内使用。在4孔培养皿的孔中分配500μL，在38.5°C的5％CO 2的潮湿空气中孵育至少2小时。 </li><li>从≥5和≤25毫米卵泡中取出COC后，将其放入装有2 mL HEPES-TCM199的培养皿（直径3.5厘米）。将COC移至盘的不同区域，以清洗细胞碎片。 </li><li>将COC转移到IVM培养基中，在38.5℃的5％CO 2的加湿空气中孵育28小时。 </li></ol></li></ol> 2.免疫荧光和图像分析<ol><li> 染色体数 <ol><li>从优势卵泡或IVM结束收集后，在38.5°C的5％CO 2的潮湿空气中孵育具有100μMMonastrol的COC 1小时。预先准备monastrol作为100x股票，并将其存储在-20°C长达1年。 </li><li>去除卵丘细胞通过用0.5％透明质酸酶或轻轻移液处理它们;通过在0.2％的链霉蛋白酶处理它去除透明带。预先将透明质酸酶和链霉蛋白酶预先作为10x储备，并将其储存在-20°C长达1年。 </li><li>将DPBS中的4％多聚甲醛中的卵母细胞在38.5℃下固定15分钟，然后在4℃下固定45分钟。 警告！处理多聚甲醛时请戴上个人防护装备，并按照危险废物处置指南处理受污染的物质。 </li><li>通过在填充有500μL补充有0.1％聚乙烯醇（PVA）的DPBS的3孔中顺序转移来洗涤卵母细胞。在室温（RT）下，在0.3％Triton X-100中渗透10分钟。 </li><li>在加入1％牛血清白蛋白（DPBS-1％BSA）和10％正常驴血清的DPBS中，在室温下封闭非特异性结合孵育至少30分钟。样品可以在4℃下保持封闭溶液3-4天。 </li><li>对于原代染色，在4℃下补充有1％BSA（稀释倍数1:50）的DPBS中，将兔抗Aurora B磷酸-323-2卵母细胞孵育过夜。 </li><li>如步骤2.1.4所述洗涤卵母细胞。将卵母细胞在黑色中在室温下在异硫氰酸四甲基罗丹明（TRITC） - 缀合的驴抗兔IgG（DPBS-1％BSA中1:100）的溶液中孵育1小时。 </li><li>按步骤2.1.4中所述清洗卵母细胞，然后将3-4个卵母细胞放入一滴不含硬化的抗褪色载体中，补充有20μMYOPRO1载玻片。重复上述步骤，直到所有的卵母细胞被安装（每个载玻片3-4个卵母细胞）。 </li><li>让卵母细胞在载物介质中沉降约10分钟，然后用盖玻片盖住它们。为了避免粉碎卵母细胞，在将玻片放在玻璃片上之前应用两条双面胶带。 注意:此预防措施还将确保所有样品在玻璃are之间均匀压缩de和盖玻片。玻璃载玻片可以在-20℃冷冻，并在随后的2-3天内成像。 </li><li>使用红色（着丝粒）和绿色（染色体）通道7,13,14,20,21，用60X物镜在共聚焦激光扫描显微镜上成像样品。在z轴上扫描整个异位板的样品，每0.35μm的步长。保存每个计划的数字化图像。 </li><li>使用NIH ImageJ软件的细胞计数功能（可从https://imagej.nih.gov/ij/plugins/cell-counter.html获得）计数串联共焦区域中的着丝点。 注意:避免重复计数出现在相邻部分上的着丝粒，通过识别出现，停留和使用数字代码在连续部分消失的着丝粒。 </li><li>重复染色体共在步骤2.1.11中与独立操作员进行通信。 注意:将卵母细胞分类为整倍体（32条染色体），超倍体（&gt; 32）或次倍体（&lt;32）。 </li></ol></li><li> 主轴形态和组蛋白乙酰化 <ol><li>从优势卵泡或IVM结束后收集，通过在0.5％透明质酸酶处理或通过温和移液去除卵丘细胞，如步骤2.1.2所述。 </li><li>洗涤DPBS-PVA中的卵母细胞，如步骤2.1.4，并在38℃下将它们固定在2.5％多聚甲醛中20分钟。如步骤2.1.4所述进行广泛洗涤，并在室温下在0.1％Triton X-100中透化5分钟。 注意:处理多聚甲醛时应戴上个人防护装备，并按照危险废物处理指南处理受污染的物质。 </li><li>在补充有2％牛血清白蛋白（DPBS-2％BSA），0.05％皂苷和10％正常驴血清的DPBS中阻断非特异性结合孵育r 2 h。 注意:样品可以在4℃下保存在封闭溶液中3-4天。 </li><li>对于原代染色，在含有0.05％皂角苷的DPBS-2％BSA中，在4℃孵育小鼠抗-α-微管蛋白（1:150）中的卵母细胞和兔抗acH4K16（1:250）过夜。 </li><li>洗涤卵母细胞如步骤2.1.4。在含有0.05％皂苷的DPBS-2％BSA中的绿色荧光染料缀合的驴抗小鼠IgG（1:500）和TRITC缀合的驴抗兔IgG（1:100）的溶液中孵育卵母细胞1小时RT在黑暗中。 </li><li>按步骤2.1.4清洗卵母细胞，然后将3-4个卵母细胞置于一滴补充有1μg/ mL 4&#39;，6-二脒基-2-苯基吲哚（DAPI）的非硬化抗褪色载体中，在玻璃片上。 </li><li>将载玻片上的卵母细胞载于步骤2.1.8。 </li><li>使用红色（acH4K16），绿色（α-微管蛋白）和蓝色（DNA）通道的60x物镜在共聚焦激光扫描显微镜上成像样品。 注意:在采集所有样品时，请保持红色和蓝色通道的激光设置不变。扫描整个减数分裂主轴和中间板在z轴上的样品，每0.35微米的步长。保存数字化图像（单图和投影3D图像）。 </li><li>使用NIH ImageJ软件的测量功能（https://imagej.nih.gov/ij/docs/guide/146-30）测量投影图像上最大宽度的极点主轴长度和主轴直径的.html）。重复3次，计算平均值。 </li><li>使用NIH ImageJ软件的集成密度函数（https://imagej.nih.gov/ij/docs/guide/146-30.html）量化acH4K16的相对荧光。将其归一化为DAPI染色的整合密度。 </li></ol></li></ol> 3. mRNA表达分析<ol><li>在从5至25毫米卵泡中取出COCs之后，收集颗粒细胞维持在培养皿中，并在冷DPBS中以10,600 xg离心2分钟洗涤两次。在液氮中快速冻结。将样品储存在-80°C长达6个月;细胞将用于标准曲线。 </li><li>仔细地从未成熟（GV）， 体外成熟和体内成熟的卵母细胞中去除所有卵丘细胞，如步骤2.1.2所述。 </li><li>洗涤卵母细胞，如2.1.4所述，在DPBS-PVA中，以1μL的体积单独收集，并在上面放置2μLRNALater。在液氮中快速冷冻。将样品储存在-80°C达6个月。 </li><li>向每个样品添加2 pg 萤光素酶RNA作为穗状。将卵母细胞2×2收集，并使用适合从小样品回收RNA的基于二氧化硅膜滤器的试剂盒提取总RNA。 </li><li>用0.25μg随机六聚体和小鼠Moloney白血病病毒逆转录酶在10μL反转录RNA 1 h，37＆＃<html> 176℃。将样品在-20°C下储存长达6个月。 </li><li>将无RNAse水的颗粒细胞中的cDNA稀释至50 ng /μL。连续稀释该溶液1:10，得到5 ng /μL，0.5 ng /μL，0.05 ng /μL和0.005 ng /μL溶液。 </li><li>使用相当于0.05个卵母细胞作为底物的cDNA，或者5μL连续稀释的颗粒细胞cDNA，10μL的SYBR绿色超级细胞和0.3μM的每种特异性引物（每个反应物表1 ）。 </li><li>使用3步方案在热循环仪中运行反应:95℃30秒，60℃30秒，72℃20秒，重复40次。最后，从60°C开始，获得熔融曲线，每20s高达95°C将温度提高0.5°C。 </li><li>使用标准曲线评估卵母细胞样品中特异性mRNA的起始量（SQ）基于颗粒细胞样品计算，将数据表示为感兴趣基因的SQ和持家基因的SQ之间的比例。 </li></ol>

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H., Hochi, S., Braun, J., Sato, K., Oguri, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+maturation+of+equine+oocytes+collected+by+follicle+aspiration+and+by+the+slicing+of+ovaries.">In vitro maturation of equine oocytes collected by follicle aspiration and by the slicing of ovaries.</a> Theriogenology. 40 (5), 959-966 (1993).</li><li>Hassold, T., Hunt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+err+(meiotically)+is+human:+the+genesis+of+human+aneuploidy.">To err (meiotically) is human: the genesis of human aneuploidy.</a> Nat Rev Genet. 2 (4), 280-291 (2001).</li><li>Akiyama, T., Nagata, M., Aoki, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inadequate+histone+deacetylation+during+oocyte+meiosis+causes+aneuploidy+and+embryo+death+in+mice.">Inadequate histone deacetylation during oocyte meiosis causes aneuploidy and embryo death in mice.</a> Proc Natl Acad Sci U S A. 103 (19), 7339-7344 (2006).</li><li>Homer, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mad2+prevents+aneuploidy+and+premature+proteolysis+of+cyclin+B+and+securin+during+meiosis+I+in+mouse+oocytes.">Mad2 prevents aneuploidy and premature proteolysis of cyclin B and securin during meiosis I in mouse oocytes.</a> Genes Dev. 19 (2), 202-207 (2005).</li><li>Rambags, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numerical+chromosomal+abnormalities+in+equine+embryos+produced+in+vivo+and+in+vitro.">Numerical chromosomal abnormalities in equine embryos produced in vivo and in vitro.</a> Mol Reprod Dev. 72 (1), 77-87 (2005).</li><li>Nabti, I., Marangos, P., Bormann, J., Kudo, N. R., Carroll, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-mode+regulation+of+the+APC/C+by+CDK1+and+MAPK+controls+meiosis+I+progression+and+fidelity.">Dual-mode regulation of the APC/C by CDK1 and MAPK controls meiosis I progression and fidelity.</a> J Cell Biol. 204 (6), 891-900 (2014).</li><li>Shomper, M., Lappa, C., FitzHarris, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetochore+microtubule+establishment+is+defective+in+oocytes+from+aged+mice.">Kinetochore microtubule establishment is defective in oocytes from aged mice.</a> Cell Cycle. 13 (7), 1171-1179 (2014).</li><li>Luzzo, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+fat+diet+induced+developmental+defects+in+the+mouse:+oocyte+meiotic+aneuploidy+and+fetal+growth+retardation/brain+defects.">High fat diet induced developmental defects in the mouse: oocyte meiotic aneuploidy and fetal growth retardation/brain defects.</a> PLoS One. 7 (11), e49217 (2012).</li><li>Ma, P., Schultz, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+deacetylase+2+(HDAC2)+regulates+chromosome+segregation+and+kinetochore+function+via+H4K16+deacetylation+during+oocyte+maturation+in+mouse.">Histone deacetylase 2 (HDAC2) regulates chromosome segregation and kinetochore function via H4K16 deacetylation during oocyte maturation in mouse.</a> PLoS Genet. 9 (3), e1003377 (2013).</li><li>Yang, F., Baumann, C., Viveiros, M. M., De La Fuente, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+hyperacetylation+during+meiosis+interferes+with+large-scale+chromatin+remodeling,+axial+chromatid+condensation+and+sister+chromatid+separation+in+the+mammalian+oocyte.">Histone hyperacetylation during meiosis interferes with large-scale chromatin remodeling, axial chromatid condensation and sister chromatid separation in the mammalian oocyte.</a> Int J Dev Biol. 56 (10-12), 889-899 (2012).</li><li>Luciano, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocytes+isolated+from+dairy+cows+with+reduced+ovarian+reserve+have+a+high+frequency+of+aneuploidy+and+alterations+in+the+localization+of+progesterone+receptor+membrane+component+1+and+aurora+kinase+B.">Oocytes isolated from dairy cows with reduced ovarian reserve have a high frequency of aneuploidy and alterations in the localization of progesterone receptor membrane component 1 and aurora kinase B.</a> Biol Reprod. 88 (3), 58 (2013).</li><li>Luciano, A. M., Lodde, V., Franciosi, F., Ceciliani, F., Peluso, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progesterone+receptor+membrane+component+1+expression+and+putative+function+in+bovine+oocyte+maturation,+fertilization,+and+early+embryonic+development.">Progesterone receptor membrane component 1 expression and putative function in bovine oocyte maturation, fertilization, and early embryonic development.</a> Reproduction. 140 (5), 663-672 (2010).</li><li>Terzaghi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PGRMC1+participates+in+late+events+of+bovine+granulosa+cells+mitosis+and+oocyte+meiosis.">PGRMC1 participates in late events of bovine granulosa cells mitosis and oocyte meiosis.</a> Cell Cycle. , 1-14 (2016).</li><li>Susor, A., Jansova, D., Anger, M., Kubelka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translation+in+the+mammalian+oocyte+in+space+and+time.">Translation in the mammalian oocyte in space and time.</a> Cell Tissue Res. 363 (1), 69-84 (2016).</li><li>Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+translation+reveals+a+critical+role+for+deleted+in+azoospermia-like+(Dazl)+at+the+oocyte-to-zygote+transition.">Genome-wide analysis of translation reveals a critical role for deleted in azoospermia-like (Dazl) at the oocyte-to-zygote transition.</a> Genes Dev. 25 (7), 755-766 (2011).</li><li>Ma, J., Flemr, M., Strnad, H., Svoboda, P., Schultz, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternally+recruited+DCP1A+and+DCP2+contribute+to+messenger+RNA+degradation+during+oocyte+maturation+and+genome+activation+in+mouse.">Maternally recruited DCP1A and DCP2 contribute to messenger RNA degradation during oocyte maturation and genome activation in mouse.</a> Biol Reprod. 88 (1), 11 (2013).</li></ol>

作者没有什么可以披露的。

即使IVM已经在马上进行了超过二十年16 ，我们还不知道卵母细胞是否可以是胚胎非整倍体的起源，正如人类提出的一样。原因可能是卵母细胞扩散用于染色体计数导致相当大的样品损失。考虑到这一点，进行了调查染色体分离错误的方法的调查，以寻找适用于马卵母细胞的最合适的技术。在科学文献中发现了四种主要技术:1）染色体扩增5,18,19,2）荧光原位杂交（FISH）4,6,17,...

已经开发了广泛的辅助生殖技术来治疗妇女，伴侣动物和濒危物种的不育症。临床设置中最常见的手术之一是通过超声引导经阴道抽吸，卵子吸收（OPU） 1从卵巢卵泡中检索中期II（MII）阶段卵母细胞。然后将这些卵母细胞体外受精 （IVF），将所得的胚胎植入受体子宫。在外源性促性腺激素给药后，取出MII期（成熟）卵母细胞。然而，这种治疗在一些患者中与卵巢过度刺激综合征（OHSS）的发展有关2 。 利用完全成熟的未成熟卵母细胞（GV-stage）的内在能力自发恢复减数分裂，从卵泡中分离出来，可以获得成熟的卵母细胞，而不需要管理调理促性腺激素3 。该过程称为卵母细胞体外成熟（IVM），并且代表了辅助生殖技术的药物导向较少，成本更低且更耐用的方法。然而，胚胎发育与体外成熟卵母细胞的成功通常低于体内成熟卵母细胞4,5 。可能的解释是， 体外成熟的卵母细胞更多地受到染色体分离中的错误的影响，并且由此产生的非整倍体损害了正常的胚胎发育6 。 了解IVM期间染色体错分离的分子基础将最终披露这种技术的全部潜力。在这方面，实验方法用于研究体外成熟卵母细胞的形态和生化特征， 与体内成熟相比这里描述了卵母细胞7,8 。具体来说，使用成年和自然循环马作为实验模型说明未成熟卵母细胞的OPU和未成熟卵母细胞的IVM的程序。然后，使用免疫荧光和图像分析来研究染色体分离，纺锤形态和组蛋白乙酰化对这些配子的全局模式。最后，描述了用于mRNA表达分析的逆转录和定量PCR方案。 与啮齿动物模型相比，马不允许遗传操作，操作不太方便，需要昂贵的维护。然而，由于与人类卵巢生理学的相似性，这种模型对于卵母细胞成熟的研究正在获得相当大的兴趣，/ SUP&gt;。此外，在马中制定IVM-IVF的可靠方案具有重大的经济利益，因为它将允许从遗传价值高的母马的数量增加。 对卵母细胞进行实验的一个局限性，特别是在单调节物种中，是限制样品的可用性。通过将以前在小鼠中开发的方法调整到马卵母细胞13,14以便进行最小化样品损失的染色体计数（参见与其他可用技术的比较的讨论），已经克服了这个限制。此外，已经优化了三重荧光染色方案以对相同的样品进行多重分析，并且仅在2个卵母细胞池上进行q-PCR分析。 总体而言，本研究描述了一种旨在形态和生物化学方面的实验方法使马卵母细胞变得更细，这种细胞类型由于样本可用性低而极具挑战性。然而，它可以扩大我们对生殖生物学和不育性不育的知识。

<table><tbody><tr><td>ultrasound probe</td><td>Aloka</td><td>UST-5820-7,5</td><td></td></tr><tr><td>human chorionic gonadotrophin</td><td>centravet</td><td>CHO004</td><td>1,500 unit/animal IV</td></tr><tr><td>detomidine</td><td>centravet</td><td>MED010</td><td>9 - 15 &amp;micro;g/kg IV</td></tr><tr><td>butylscopolamine bromure</td><td>centravet</td><td>EST001</td><td>0.2 mg/kg IV</td></tr><tr><td>stereomicroscope</td><td>NIKON</td><td>SMZ-2B</td><td></td></tr><tr><td>butorphanol</td><td>centravet</td><td>DOL003</td><td>10&amp;micro;g/kg IV</td></tr><tr><td>benzyl-penicillin</td><td>centravet</td><td>DEP203 IM</td><td>15,000 UI/animal</td></tr><tr><td>TCM199</td><td>Sigma-Aldrich</td><td>M3769 10X1L</td><td>powder for hepes-buffered TCM199</td></tr><tr><td>Hepes sodium salt</td><td>Sigma-Aldrich</td><td>H3784-100G</td><td></td></tr><tr><td>bovine serum albumin</td><td>Sigma-Aldrich</td><td>A8806</td><td></td></tr><tr><td>heparin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>H3149-10KU</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;-buffered TCM199</td><td>Sigma-Aldrich</td><td>M2154-500ML</td><td>liquid for IVM medium</td></tr><tr><td>epidermal growth factor</td><td>Sigma-Aldrich</td><td>E4127</td><td></td></tr><tr><td>newborn calf serum</td><td>Sigma-Aldrich</td><td>N4762-500ML</td><td></td></tr><tr><td>4-well dishes</td><td>NUNCLON</td><td>144444</td><td></td></tr><tr><td>incubator</td><td>Heraeus</td><td>BB6060</td><td></td></tr><tr><td>monastrol</td><td>Sigma-Aldrich</td><td>M8515</td><td></td></tr><tr><td>hyaluronidase</td><td>Sigma-Aldrich</td><td>H3506</td><td></td></tr><tr><td>pronase</td><td>Sigma-Aldrich</td><td>P5147</td><td></td></tr><tr><td>paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>polyvinyl alcohol</td><td>Sigma-Aldrich</td><td>341584</td><td></td></tr><tr><td>triton-X 100</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>normal donkey serum</td><td>Sigma-Aldrich</td><td>D9663</td><td></td></tr><tr><td>rabbit anti-Aurora B phospho-Thr232</td><td>BioLegend</td><td>636102</td><td></td></tr><tr><td>TRITC-conjugated donkey anti-rabbit IgG</td><td>Vector Laboratories</td><td>711-025-152</td><td></td></tr><tr><td>Vecta-Shield</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>YOPRO1</td><td>Thermofisher Scientific</td><td>Y3603</td><td></td></tr><tr><td>confocal laser scanning microscope</td><td>LSM 780</td><td>Zeiss</td><td></td></tr><tr><td>confocal laser scanning microscope</td><td>LSM 700</td><td>Zeiss</td><td></td></tr><tr><td>ImageJ software</td><td>rsb.info. nih.gov/ij/download.html</td><td>free resource</td><td></td></tr><tr><td>mouse anti-alpha-tubulin</td><td>Sigma-Aldrich</td><td>T8203</td><td></td></tr><tr><td>rabbit anti-acH4K16</td><td>Upstate Biotechnology</td><td>07-329</td><td></td></tr><tr><td>AlexaFluor 488-coniugated donkey anti-mouse IgG</td><td>Life Technologies</td><td>A21202</td><td></td></tr><tr><td>4&amp;rsquo;,6-diamidino-2-phenylindole</td><td>Sigma-Aldrich</td><td>D8417</td><td>DAPI</td></tr><tr><td>centrifuge</td><td>Eppendorf</td><td>5417R</td><td></td></tr><tr><td>RNALater</td><td>Invitrogen</td><td>AM7020</td><td></td></tr><tr><td>Luciferase RNA</td><td>Promega</td><td>L4561</td><td></td></tr><tr><td>PicoPure RNA Isolation Kit</td><td>Applied Biosystems</td><td>12204-01</td><td></td></tr><tr><td>random hexamers</td><td>Thermofisher Scientific</td><td>N8080127</td><td></td></tr><tr><td>mouse Moloney leukaemia virus reverse transcriptase</td><td>Thermofisher Scientific</td><td>28025013</td><td></td></tr><tr><td>SYBR green supermix</td><td>BioRad</td><td>1708880</td><td></td></tr><tr><td>specific primers</td><td>Sigma-Aldrich</td><td></td><td>specific primers were designed using Primer3Plus software (free resource)</td></tr><tr><td>thermal-cycler</td><td>BioRad</td><td>MyiQ</td><td></td></tr><tr><td>mouse monoclonal anti-CENPA</td><td>Abcam</td><td>ab13939</td><td></td></tr><tr><td>mouse monoclonal anti-Aurora B</td><td>Abcam</td><td>ab3609</td><td></td></tr></tbody></table>

analysis of chromosome segregation, histone acetylation, and spindle morphology in horse oocytes

辅助生殖领域已被开发用于治疗妇女，伴侣动物和濒危物种的不育症。在马上，辅助生殖也允许从高绩效者生产胚胎，而不会中断他们的运动生涯，并有助于增加从高遗传价值的母马的数量。本手稿描述了使用卵子吸收（OPU）从马卵巢收集未成熟和成熟卵母细胞的程序。然后通过适应以前在小鼠中发展的方案，将这些卵母细胞用于研究非整倍体的发生率。具体而言，共焦激光显微镜扫描后，染色体和中期II（MII）卵母细胞的着丝粒被荧光标记并计数在连续的焦点图上。该分析显示，当从卵泡中收集未成熟卵母细胞并在体外成熟时，非整倍体率的发生率较高在体内。微管蛋白的免疫染色和特定赖氨酸残基的组蛋白4的乙酰化形式也揭示了减数分裂纺锤体的形态和组蛋白乙酰化的全局模式的差异。最后，通过逆转录和定量PCR（q-PCR）研究了编码组蛋白脱乙酰酶（HDAC）和乙酰转移酶（HAT）的mRNA的表达。 在体外和体内成熟的卵母细胞之间没有观察到转录物的相对表达的差异。与卵母细胞成熟期间转录活性的一般沉默一致，总转录物量的分析只能揭示mRNA的稳定性或降解。因此，这些研究结果表明，其他翻译和翻译后法规可能会受到影响。 总体而言，本研究描述了一种用于形态和生物化学表征马卵母细胞的实验方法由于样品可用性低，这种类型的研究极具挑战性。然而，它可以扩大我们对单调繁殖生物学和不孕症的知识。

这些实验的原始发现在以前已经深入描述7并且在本文中作为可以使用所述方案获得的结果的实例进行报道。 成熟率在OPU从优势卵泡获得的32个COCs中，28个（88％）处于MII阶段。在5-25mm大小的卵泡中收集的58个COC中的14个被立即快速冷冻为未成熟的卵母细胞用于mRNA表达...

Watch this Scientific Journal Video about Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes at JoVE.com

马卵母细胞染色体分离、组蛋白乙酰化和纺锤体形态分析- 中国优秀硕士学位论文全文数据库发育生物学|视频

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

该手稿描述了一种用于形态和生物化学表征马卵母细胞的实验方法。具体来说，本文通过超声引导的卵子吸收（OPU）如何收集不成熟和成熟的马卵母细胞，以及如何调查染色体分离，纺锤形态，全局组蛋白乙酰化和mRNA表达。

马卵母细胞染色体分离，组蛋白乙酰化和主轴形态分析

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted ...

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10.9K 次观看.  University of Milan. 该手稿描述了一种用于形态和生物化学表征马卵母细胞的实验方法。具体来说，本文通过超声引导的卵子吸收（OPU）如何收集不成熟和成熟的马卵母细胞，以及如何调查染色体分离，纺锤形态，全局组蛋白乙酰化和mRNA表达。 

视频: Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

从前期到中期二期小鼠卵母细胞进展的染色质传播制剂分析

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for ...

科研

遗传学

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded Nucleolus) or unable (NSN, Not Surrounded Nucleolus) to develop to the blastocyst stage after in vitro maturation (IVM) and in vitro fertilization (IVF). It makes use of Hoeschst33342 (or any other DNA intercalating dye) able to bind to the heterochromatin of the nucleolus showing a ring in the SN oocytes or not, like in the NSN oocytes. This represents the easiest and quickest way to sort both antral oocytes that can be eventually used for IVM or IVF procedures. 
Briefly, the protocol consists of the following steps: hormone injection to stimulate follicular growth; isolation of the oocytes at the GV stage from the antral compartment by puncturing the ovary with a sterile needle; preparation of thin glass pipettes for mouth pipetting of the oocytes; sorting of the oocytes with Hoechst33342 prepared at a supravital concentration; IVM, IVF or any other molecular/cellular analysis.
Unfortunately there are still few evidences to sort SN and NSN oocytes using less invasive techniques. If and once they will be identified, they could be potentially applied to human assisted reproductive technologies, although with several aspects that should be modified. To date, this technique has potential implications to dramatically increase IVM and IVF successful procedures in both endangered and species with economic interest.

Here we present a protocol for the characterization of mouse antral oocytes based on nucleolar organization.

基于核仁染色质组织分离鼠标窦卵母细胞与表征

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

发育生物学

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

小鼠卵母细胞在减数分裂过程中对人类基因功能的评估

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Human Egg Maturity Assessment and Its Clinical Application

The optimal timing of intracytoplasmic sperm injection (ICSI) is of a serious concern for fertility programs because untimely sperm entry diminishes the egg&#39;s developmental competence. Presence of the first polar body (PB) together with the meiotic spindle indicates completion of the oocyte maturation and the egg&#39;s readiness for fertilization. In clinical practice, it is customary to assume that all oocytes displaying a PB are mature metaphase (MII) oocytes. However, PB extrusion precedes the formation of the bipolar MII spindle. This asynchrony makes the mere presence of PB an unreliable marker of oocyte maturity. Noninvasive spindle imaging using polarized light microscopy (PLM) allows quick and easy inspection of whether the PB-displaying oocyte actually reassembled a meiotic spindle prior to ICSI. Here, we present a standard protocol to perform human egg maturity assessment in the clinical laboratory. We also show how to optimize the time of ICSI with respect to the oocyte&#39;s developmental stage in order to prevent premature sperm injection of late-maturing oocytes. Using this approach, even immature oocytes extruding PB in vitro can be clinically utilized. Affirmation that MII spindle is present prior to sperm injection and individual adjustment of the time of ICSI is particularly important in poor prognosis in-vitro fertilization (IVF) cycles with a low number of oocytes available for fertilization.

We provide an outline of the clinical protocol for non-invasive assessment of human egg maturity using polarized light microscopy.

人卵成熟度评估及其临床应用

The optimal timing of intracytoplasmic sperm injection (ICSI) is of a serious concern for fertility programs because untimely sperm entry diminishes the ...

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give rise to aneuploidy occur during meiosis in oocytes, but why oocyte meiosis is error-prone is still not fully understood. During cell division, cells prevent errors in chromosome segregation by activating the spindle assembly checkpoint (SAC). This control mechanism relies on detecting kinetochore (KT)-microtubule (MT) attachments and sensing tension generated by spindle fibers. When KTs are unattached, the SAC is activated and prevents cell-cycle progression. The SAC is activated first by MPS1 kinase, which triggers the recruitment and formation of the mitotic checkpoint complex (MCC), composed of MAD1, MAD2, BUB3, and BUBR1. Then, the MCC diffuses into the cytoplasm and sequesters CDC20, an anaphase-promoting complex/cyclosome (APC/C) activator. Once KTs become attached to microtubules and chromosomes are aligned at the metaphase plate, the SAC is silenced, CDC20 is released, and the APC/C is activated, triggering the degradation of Cyclin B and Securin, thereby allowing anaphase onset. Compared to somatic cells, the SAC in oocytes is not as effective because cells can undergo anaphase despite having unattached KTs. Understanding why the SAC is more permissive and if this permissiveness is one of the causes of chromosome segregation errors in oocytes still needs further investigation. The present protocol describes the three techniques to comprehensively evaluate SAC integrity in mouse oocytes. These techniques include using nocodazole to depolymerize MTs to evaluate the SAC response, tracking SAC silencing by following the kinetics of Securin destruction, and evaluating the recruitment of MAD2 to KTs by immunofluorescence. Together these techniques probe mechanisms needed to produce healthy eggs by providing a complete evaluation of SAC integrity.

Error in chromosome segregation is a common feature in oocytes. Therefore, studying the spindle assembly checkpoint gives important clues about the mechanisms needed to produce healthy eggs. The present protocol describes three complementary assays to evaluate spindle assembly checkpoint integrity in mouse oocytes.

评估小鼠卵母细胞中纺锤体组件检查点完整性

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give ...

Multi-Photon Laser Ablation of Cytoplasmic Microtubule Organizing Centers in Mouse Oocytes

The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at prophase I of the first meiotic division. After puberty and upon meiotic resumption, the nuclear membrane disassembles (nuclear envelope breakdown), and the spindle is assembled mainly at the oocyte center. Initial central spindle positioning is essential to protect against abnormal kinetochore-microtubule (MT) attachments and aneuploidy. The centrally positioned spindle migrates in a time-sensitive manner toward the cortex, and this is a necessary process to extrude a tiny polar body. In mitotic cells, spindle positioning relies on the interaction between centrosome-mediated astral MTs and the cell cortex. On the contrary, mouse oocytes lack classic centrosomes and, instead, contain numerous acentriolar MT organizing centers (MTOCs). At the metaphase I stage, mouse oocytes have two different sets of MTOCs: (1) MTOCs that are clustered and sorted to assemble spindle poles (polar MTOCs), and (2) metaphase cytoplasmic MTOCs (mcMTOCs) that remain in the cytoplasm and do not contribute directly to spindle formation but play a crucial role in regulating spindle positioning and timely spindle migration. Here, a multi-photon laser ablation method is described to selectively deplete endogenously labeled mcMTOCs in oocytes collected from Cep192-eGfp reporter mice. This method contributes to the understanding of the molecular mechanisms underlying spindle positioning and migration in mammalian oocytes.

An optimized protocol is presented that enables the depletion of cytoplasmic microtubule organizing centers in mouse oocytes during metaphase I using a near-infrared femtosecond laser.

小鼠卵母细胞质微管组织中心的多光子激光消融

The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at ...

鉴定小鼠前期停滞卵母细胞中的 DNA 损伤

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the prophase of meiosis I. The quiescent state may last for years until the oocytes receive a stimulus to grow and obtain the competency to resume meiosis. This protracted state of arrest makes them extremely susceptible to accumulating DNA-damaging insults, which affect the genetic integrity of the female gametes and, therefore, the genetic integrity of the future embryo. 
Consequently, the development of an accurate method to detect DNA damage, which is the first step for the establishment of DNA damage response mechanisms, is of vital importance. This paper describes a common protocol to test the presence and progress of DNA damage in prophase-arrested oocytes during a period of 20 h. Specifically, we dissect mouse ovaries, retrieve the cumulus-oocyte complexes (COCs), remove the cumulus cells from the COCs, and culture the oocytes in &#924;2 medium containing 3-isobutyl-1-methylxanthine to maintain the state of arrest. Thereafter, the oocytes are treated with the cytotoxic, antineoplasmic drug, etoposide, to engender double-strand breaks (DSBs). 
By using immunofluorescence and confocal microscopy, we detect and quantify the levels of the core protein &#947;H2AX, which is the phosphorylated form of the histone H2AX. H2AX becomes phosphorylated at the sites of DSBs after DNA damage. The inability to restore DNA integrity following DNA damage in oocytes can lead to infertility, birth defects, and increased rates of spontaneous abortions. Therefore, the understanding of DNA damage response mechanisms and, at the same time, the establishment of an intact method for studying these mechanisms are essential for reproductive biology research.

作者聚焦：了解哺乳动物卵母细胞和植入前胚胎的 DNA 损伤反应 

Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ cells.

检测小鼠卵母细胞中的DNA双链断裂

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

小鼠卵母细胞显微注射，成熟和倍性评估

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

马卵母细胞染色体分离，组蛋白乙酰化和主轴形态分析

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此视频中的章节

从前期到中期二期小鼠卵母细胞进展的染色质传播制剂分析

基于核仁染色质组织分离鼠标窦卵母细胞与表征

小鼠卵母细胞在减数分裂过程中对人类基因功能的评估

人卵成熟度评估及其临床应用

评估小鼠卵母细胞中纺锤体组件检查点完整性

小鼠卵母细胞质微管组织中心的多光子激光消融

检测小鼠卵母细胞中的DNA双链断裂

检测小鼠卵母细胞中的DNA双链断裂

检测小鼠卵母细胞中的DNA双链断裂

小鼠卵母细胞显微注射，成熟和倍性评估