The Effect of Vitrification on Mouse Oocyte Apoptosis by Cryotop Method

Background : Oocyte cryopreservation is one of the most important topics in the field of assisted reproductive technology to preserve women fertility, but relationship between cryopreservation and apoptosis is still a matter of debate. The present study was aimed to investigate the effects of vitrification on apoptosis in mouse oocytes by Cryotop method. Method: A total of 200 germinal vesicle (GV) and 200 metaphase II (MII) oocytes were obtained from ovaries and fallopian tubes of NMRI mice, respectively and divided into control and experimental groups. Oocytes in experimental group were vitrified by Cryotop using vitrification medium and were kept in liquid nitrogen for one month. The survival rate of oocytes was evaluated after 2 hour incubation time. Then, the oocyte apoptosis was evaluated by TUNEL technique and compared with those in control group. The data was compared statistically using SPSS software and chi-square test. Results: The survival rates of vitrified GV (93%) and MII oocytes (88%) showed a significant decrease compared with the control group (P<0.05), but there was no significant difference in survival rate of both vitrified oocyte groups. The incidence of apoptosis in vitrified and control GV oocytes showed no significant difference (13% vs. 7%), but the rate of apoptosis in vitrified MII oocytes increased significantly not only in comparison with MII control group (25% vs. 5%) but also with vitrified GV oocytes (P<0.05). Conclusion: The results indicate that vitrification increases apoptosis in mouse MII oocytes and apoptosis may play a role in MII oocyte injury after vitrification. Key Words: Vitrification, Apoptosis, Oocytes     

Distinct functions of <Emphasis Type="Italic">S. pombe</Emphasis>Rec12 (Spo11) protein and Rec12-dependent crossover recombination (chiasmata) in meiosis I; and a requirement for Rec12 in meiosis II

BACKGROUND: In most organisms proper reductional chromosome segregation during meiosis I is strongly correlated with the presence of crossover recombination structures (chiasmata); recombination deficient mutants lack crossovers and suffer meiosis I nondisjunction. We report that these functions are separable in the fission yeast Schizosaccharomyces pombe. RESULTS: Intron mapping and expression studies confirmed that Rec12 is a member of the Spo11/Top6A topoisomerase family required for the formation of meiotic dsDNA breaks and recombination. rec12-117, rec12-D15 (null), and rec12-Y98F (active site) mutants lacked most crossover recombination and chromosomes segregated abnormally to generate aneuploid meiotic products. Since S. pombe contains only three chromosome pairs, many of those aneuploid products were viable. The types of aberrant chromosome segregation were inferred from the inheritance patterns of centromere linked markers in diploid meiotic products. The rec12-117 and rec12-D15 mutants manifest segregation errors during both meiosis I and meiosis II. Remarkably, the rec12-Y98F (active site) mutant exhibited essentially normal meiosis I segregation patterns, but still exhibited meiosis II segregation errors. CONCLUSIONS: Rec12 is a 345 amino acid protein required for most crossover recombination and for chiasmatic segregation of chromosomes during meiosis I. Rec12 also participates in a backup distributive (achiasmatic) system of chromosome segregation during meiosis I. In addition, catalytically-active Rec12 mediates some signal that is required for faithful equational segregation of chromosomes during meiosis II.     

Molecular Cytogenetic Analysis of Spontaneous Interspecific Hybrid Between Oryza sativa and Oryza minuta

Genomic in situ hybridization (GISH) is a powerful tool to characterize parental chromosomes in interspecific hybrids, including the behaviour of autosynapsis and chromosome paidng. It was used to distinguish the chromosomes of Oryza sativa from wild species in a spontaneous interspecific hybdd and to investigate the chromosome pairing at metaphase I in meiosis of the hybdd in this study. The hybrid was a triploid with 36 chromosomes according to the chromosome nurnber investigated in mitosis of root tips. During metaphase I of meiosis in the hybrid, less chromosome pairing was observed and most of the chromosomes existed as univalent. Based on GISH and FISH (Fluorescent in situ hybridization) analyses, the chromosomes of the hybrid were composed of genomes A, B and C. Thus, it was believed that the hybrid was the result of natural hybridization between cultivated rice and wild species O. minuta which was planted in experimental fields.     

稻属种间天然异交种的分子细胞学鉴定

利用基因组荧光原位杂交（GISH）技术快速鉴定了栽培稻与野生稻的天然异交种的基因组组成,分析了该杂种在减数分裂中期Ⅰ的染色体配对情况。根据根尖细胞的染色体数目,发现该杂种是具有36条染色体的三倍体;通过减数分裂中期Ⅰ染色体的配对研究,发现该杂种染色体很少发生配对,绝大部分染色体以单价体形式存在;结合GISH技术的分析,证实该杂种是由A、B和C 3个染色体组组成。因此该杂种是栽培稻和小粒野生稻的天然杂交种。     

甲基氨草磷对橡胶树悬浮细胞同步化及微核诱导的影响

植物微原生质体融合(microprotoplast fusion)是将部分基因组转移而又避免染色体损伤的不对称融合技术,微原生质体成功诱导是微原生质体融合的先决条件。本研究在摸索甲基氨草磷(amiprophos-methyl,APM)对橡胶树悬浮细胞有丝分裂中期指数和染色体形态影响基础上,研究了酶解时间及酶解过程中添加APM对微原生质体产量的影响。结果发现,悬浮细胞随APM处理浓度和时间的增加,有丝分裂中期指数先升高再下降,处理12 h时达到最大值;染色体聚集程度也先增加再降低,处理16 h时染色体开始解聚。在酶解过程中添加APM和延长酶解时间会大大降低微原生质体的产量。悬浮细胞经过1 μmol/L APM处理10 h后,酶解10 h,中期细胞可达到79.4%,微核化细胞指数达到7.3%,从而建立了APM诱导橡胶树悬浮细胞同步化及高效获得橡胶树微化细胞的方法。     

新疆红花随体染色体及25S rDNA位点分析

利用荧光原位杂交技术研究我国新疆红花25S rDNA在染色体上的位点数目,同时分析了其随体染色体数目。研究结果显示：新疆红花有2对随体染色体;4对染色体存在25S rDNA位点,其中3对染色体上的杂交信号较强,可稳定检出,有时还可检测到1对染色体上较弱的杂交信号。与国外红花品种相比,新疆红花rDNA位点数目多,表明新疆红花与国外红花品种间核仁组织区结构存在较大差异。这些结果将为研究红花染色体进化及其准确的核型分析提供一定的参考。     

水稻端四体的分子细胞学鉴定及染色体行为分析

在水稻品种中籼3037第9染色体短臂端三体的自交后代中发现了一个变异株。该植株叶片内卷,株型偏散,结实率差。分子细胞学鉴定表明,该植株体细胞染色体数比正常植株多2条,多出的染色体均比较小。进一步用来源于水稻着丝粒特异DNA序列（RCS2）以及位于水稻第9染色体短臂上的特异性DNA序列为探针,进行荧光原位杂交分析,证明该变异株多出的染色体均为第9染色体短臂,故该变异株为第9染色体短臂端四体。对变异株的减数分裂染色体行为进行分析表明,在所观察的25个细胞中,96%的细胞增加的两个端着丝粒染色体可配对形成二价体,一般不与正常的第9染色体配对形成多价体。但额外染色体形成的二价体在中期Ⅰ容易发生提前解离的现象。     

45S rDNA和5S rDNA在水仙染色体上的物理定位

利用荧光原位杂交技术对水仙45S rDNA和5S rDNA进行定位分析.结果表明,45S rDNA信号共有2对,分别分布在水仙的第6号染色体和第7号染色体短臂的末端,且第7号染色体上的拷贝数多于第6号染色体的;而5S rDNA则只有l对杂交信号,位于第2号染色体的长臂,信号较弱.     

Identification of potato chromosomes with Giesma

A modified Giesma staining technique was used to identify the somatic chromosomes of diploid potatoes. Distinct banding patterns were observed on all 12 chromosomes. Individual chromosomes were identified according to the number and distribution of bands. No significant difference in banding patterns was observed between the chromosomes of Phureja-haploid Tuberosum hybrids and those ofS. chacocnse. The extra chromosome of a trisomic of 5.chacoense was identified as an isochromosome for the long arm of chromosome G.     

黑麦染色体的ASG法G—带分析

用改良 ASG法在黑麦 ( Secale cereale)有丝分裂早中期、中期对染色体进行了 G-带分析 ,并作了G-带带型和变动性分析。结果表明 ,无论是早中期还是中期 ,黑麦染色体都显示了清晰的 G-带 ,而且带纹数目多、细窄而大小较相近 ,分布较密集而均匀 ;同源染色体带纹的数目、分布位置基本一致 ,可较为准确地配对。随着分裂时期的推进 ,G-带带数减少 ,从早中期至中期单倍染色体组 G-带带数减少了 2 9.0 %,单倍染色体组的长度缩短了 2 5.0 %,带纹减少幅度与染色体长度缩短幅度相近。对黑麦 G-带的特征与变动性、G-显带技术及其应用等问题进行了讨论     

Molecular-Cytological Identification and Chromosome Behavior Analysis of Telotetrasomic in Rice

From the progenies of a telotrisomic of chromosome 9 short arm of an indica rice variety, Zhongxian 3037, a phenotypical variant was selected. The variant plant had rolled leaves, dispersed plant type, as well as a low seed-setting rate. Cytological and molecular cytological investigations revealed two extra chromosomes, which were the shortest in somatic cells of the variant. Fluorescent in situ hybridization (FISH) analysis using a rice centromere specific DNA (RCS2) and a DNA sequence specific for chromosome 9 on premetaphase and pachytene chromosomes showed that these two chromosomes were the short arms of chromosome 9. That is to say, the variant was a telotetrasomic of chromosome 9. Among the 25 pachytene cells, the two telosomic chromosomes paired each other to form a bivalent and didn't pair with other normal chromosome 9 as multivalents in 96% cells. However, the bivalent was easy to disassociate in advance.     

甜菜属近缘种三种间杂种及其后代减数分裂行为观察

本文观察了岔根甜菜(Beta Patula Ait)、叶用甜菜(B.cicla)及普通甜菜(B.vulgaris)三种间杂种的F_1、F_2及F_3的减数分裂行为。各代减数分裂终变期,染色体联会成9个二价体。中期Ⅰ有棒状结合,也有环状结合。F_1、F_2、F_3各代中期出现单价体频率分别为0.23、0.18、1.13。后期Ⅰ出现染色体桥、落后等异常现象,各代频率分别为0.93、2.2和3.26。后期Ⅱ各代异常频率为0.41、0.75和0.32。异常四分体频率各代分别为0.16、0.13和0.66。异常频率有随世代增长而增加的趋势。结果表明,B.patula、B.cicla及B.vulgaris三个种的染色体组基本上是同源的。本文还讨论了杂种减数分裂稳定性及遗传育种上利用的可能性。     

高抗白粉病和条锈病小麦-卵穗山羊草衍生后代1003的分子细胞遗传学鉴定

卵穗山羊草中蕴含着许多小麦改良所需的优良基因,是小麦重要的三级基因库。为了解其更多遗传特性,本研究利用细胞学、原位杂交、分子标记、形态学和抗病性鉴定等技术对小麦-卵穗山羊草SY159的衍生后代1003进行鉴定。细胞学鉴定结果表明,1003含有44条染色体,减数第一次分裂中期含有22个二价体且配对良好,减数第一次分裂后期含有44条染色体且均等分离;基因组原位杂交(Genomic in situ hybridization,GISH)分析显示,1003含有42条小麦染色体和2条卵穗山羊草染色体;EST和PLUG分子标记分析表明,导入的染色体属于7M染色体;荧光原位杂交(Fluorescence in situ hybridization,FISH)分析表明,1003中含有38条与中国春标准核型相一致的染色体,4A、5A和7M的FISH信号有变异;苗期抗病性鉴定结果表明,1003对白粉病生理小种E09免疫,对条锈病生理小种条中23(CYR23)高抗;形态学调查表明,1003的农艺性状介于双亲之间,千粒重高于双亲。因此,1003是一个具有白粉病和条锈病抗性的小麦-卵穗山羊草二体异附加系,可为小麦品种改良和抗病育种提供新的种质资源。     

Calyculin A,an enhancer of myosin,speeds up anaphase chromosome movement

Actin and myosin inhibitors often blocked anaphase movements in insect spermatocytes in previous experiments. Here we treat cells with an enhancer of myosin, Calyculin A, which inhibits myosin-light-chain phosphatase from dephosphorylating myosin; myosin thus is hyperactivated. Calyculin A causes anaphase crane-fly spermatocyte chromosomes to accelerate poleward; after they reach the poles they often move back toward the equator. When added during metaphase, chromosomes at anaphase move faster than normal. Calyculin A causes prometaphase chromosomes to move rapidly up and back along the spindle axis, and to rotate. Immunofluorescence staining with an antibody against phosphorylated myosin regulatory light chain (p-squash) indicated increased phosphorylation of cleavage furrow myosin compared to control cells, indicating that calyculin A indeed increased myosin phosphorylation. To test whether the Calyculin A effects are due to myosin phosphatase or to type 2 phosphatases, we treated cells with okadaic acid, which inhibits protein phosphatase 2A at concentrations similar to Calyculin A but requires much higher concentrations to inhibit myosin phosphatase. Okadaic acid had no effect on chromosome movement. Backward movements did not require myosin or actin since they were not affected by 2,3-butanedione monoxime or LatruculinB. Calyculin A affects the distribution and organization of spindle microtubules, spindle actin, cortical actin and putative spindle matrix proteins skeletor and titin, as visualized using immunofluorescence. We discuss how accelerated and backwards movements might arise.     

甘蓝型油菜-埃塞俄比亚芥种间杂交非整倍体后代的遗传

非整倍体Nj04-089为甘蓝型油菜(Brassica napus L.)与埃塞俄比亚芥(B. carinata A. Br.)种间杂交后代中获得的附加系。为分析Nj04-089的遗传特性,利用显微镜对Nj04-089自交后代的根尖细胞染色体进行计数、对花粉母细胞染色体分裂行为进行观察,并采用PCR、GISH和Southern杂交等技术对Nj04-089的2个连续自交后代的附加染色体进行了遗传分析。结果显示：这两个连续自交世代根尖细胞染色体数2n=37～51条;花粉母细胞减数分裂的中期染色体构型分别为(0-4)Ⅰ+ (16-23)Ⅱ+ (0-2)Ⅲ 和 (0-8)Ⅰ+ (14-24)Ⅱ+ (0-4)Ⅲ + (0-1)Ⅳ,且观察到染色体桥、染色体落后、分裂周期不同步等异常行为。PCR、GISH和Southern杂交结果表明在该非整倍体后代中未检测到芸薹属B组染色体或大的片段,推测非整倍体附加的染色体并非整条或大片段B组染色体。       

栽培甜菜和白花甜菜及其杂种后代染色体组型分析

利用栽培甜菜、白花甜菜及其种间杂交获得的ＶＶＣＣ、ＶＶＣ、ＶＣＣ后代有丝分裂中期细胞进行了染色体组型分析。描述了栽培甜菜、白花甜菜两个物种染色体的着丝点位置、绝对长度和相对长度，构成了它们的核型模式图。试验说明杂种细胞中各物种的染色体是稳定的，能相互比较，可以做为鉴定染色体的重要基础。尽管如此，由于甜菜染色体较小且相互相似，也存在排列顺序颠倒的危险     

水稻顶部三叶与穗重的关系及其QTL分析

 摘要: 对水稻汕优63重组自交系群体顶部3张叶片的长、宽、重和单穗重等10个性状进行了相关分析和QTL定位。穗重与9个叶片性状存在极显著的正相关，其中与倒2叶重的相关系数最大，剑叶重次之。所有性状在重组自交系群体中均存在双向超亲分离，接近正态分布。共检测到44个主效QTL和43对双位点互作影响上述10个性状。主效QTL分布于水稻的除第8染色体外的其余11条染色体上，贡献率介于3.19%~26.23%；互作分布于水稻的12条染色体上，贡献率变幅为2.03%~8.93%。第2染色体的R2510-RM211标记区间同时检测到控制单穗重和倒2叶重的QTL，该QTL对超级稻株型育种具有应用价值。     

两种水稻四体的分离和细胞学鉴定

 在中籼3037初级三体9和初级三体11的后代中分别发现了一株形态变异株。依据中籼3037减数分裂粗线期核型以及这两个变异株减数分裂粗线期、终变期及后期Ι的染色体联会特征,确定这两个变异株分别为第9染色体和第11染色体的四体。     

小麦-中间偃麦草代换系中233的分子细胞学鉴定

为了从小麦-中间偃麦草衍生后代中获得具有优良性状的新种质,利用细胞学和分子标记技术对中间偃麦草衍生系中233进行鉴定。结果表明,中233的根尖细胞染色体数为2n=42,花粉母细胞减数分裂中期Ⅰ的染色体通常配成21个二价体。以中间偃麦草基因组DNA为探针、中国春基因组DNA为封阻进行基因组原位杂交(GISH)分析发现,中233含有2条中间偃麦草染色体和40条小麦染色体,在减数分裂中期I,两条中间偃麦草染色体可以正常配对。利用D基因组特异探针pAs1进行荧光原位杂交(FISH)分析发现,中233缺少了一对小麦的2D染色体。分子标记鉴定进一步表明,中233的1对小麦2D染色体被中间偃麦草染色体所代换。说明中233是一个细胞学稳定的小麦-中间偃麦草二体代换系,初步推断其可能携带有中间偃麦草的优异基因。