牛体外成熟卵母细胞染色体形态学研究

[目的]为了给牛体细胞克隆和转基因克隆工作提供基础性材料.[方法]在显微镜下,从卵液中捡出卵丘-卵母细胞复合体,置入成熟培养液中进行成熟培养,处理培养不同时期的卵母细胞,所得的去掉卵丘的卵母细胞固定在载玻片上,染色、冲洗、晾干,在显微镜下观察.[结果]表明:成熟培养0 h到4 h大多数卵母细胞处于GV期(97.5%～87.8%),培养6 h以后GVBD发生了卵母细胞数量明显增多(51.6%),成熟培养8 h～12 h处于Pre-MI的卵母细胞逐渐减少(76.7%～43.2%),MI期卵母细胞逐渐增多(13.3%～50.0%).成熟培养16 h有17.8%的卵母细胞处于MII期,大部分细胞仍处于MI期(40.0%)和AI/TI期(42.2%).从16 h～24 h,MII期卵逐渐增多,培养24 h后大多数卵母细胞排出第一极体到达MII期(86.5%).[结论]在减数分裂过程中,染色体也发生了显著的形态变化.第一次减数分裂中期时的染色体清晰可数,进入后期时,分开的两团染色体各自凝集成染色质团,并且一直持续到末期,到达第二次减数分裂中期时又成为清晰可数的染色体状态.     

禽类染色体制备及其在育种中应用

较详细地介绍了目前常用的 4种禽类染色体的制备方法 ,同时阐述了染色体制备技术在禽类起源进化、亲缘关系鉴定、杂交不育以及种质评定等诸多方面的应用 ,并展望了染色体研究的应用前景     

黄顶菊染色体数目及核型分析(简报)

黄顶菊(Flaveria bidentis(L.)Kuntze)是最近出现在河北省衡水市衡水湖湿地国家级自然保护区、天津市、廊坊等华北地区的一种外来入侵植物,原产南美洲,后扩散到美洲中部和南部,在非洲和欧洲也有部分分布,亚洲的日本也有发现^[1]。经有关专家鉴定属于菊科堆心菊族黄顶菊属,为一年生草本,株高150cm左右,茎多汁近肉质。该种植物为蝎尾状聚伞花序,花冠黄色,产种量极高,成熟后种子为2~2.5mm黑色瘦果。高贤明等对其形态特征及生理特征做了详细介绍^[2]。     

洛阳地区八点黑獭兔染色体核型与G-带研究

采用外周血淋巴细胞培养方法,对洛阳地区八点黑獭兔的染色体核型与G-带带型进行分析。结果表明:洛阳八点黑獭兔的二倍体染色体数为2n=44,公兔的染色体核型为44,XY;母兔的染色体核型为44,XX。21对常染色体中,1～8号染色体为中着丝粒染色体,9～11号染色体为近中着丝粒染色体,12～17号染色体为近端着丝粒染色体,18～21号染色体为端着丝粒染色体。X染色体为近中着丝粒染色体,Y为最小的端着丝粒染色体。八点黑獭兔的带型基本上与其他家兔品系的染色体带型一致。     

扁穗牛鞭草染色体数目及核型分析

采用常规根尖压片法,对扁穗牛鞭草栽培品种‘广益’和野生扁穗牛鞭草材料T003染色体数目和核型进行研究,结果表明:(1)扁穗牛鞭草染色体基数x=9;(2)‘广益’扁穗牛鞭草为六倍体,核型公式为2n=6x=54=34m+10sm(2SAT)+6st+2t+2T,核不对称系数为61.43%,核型属于2B类型;(2)野生扁穗牛鞭草材料T003为四倍体,核型公式为2n=4x=36=14m+10sm(2SAT)+8st+4T,核不对称系数为65.67%,核型属于2C类型。     

陕西延安蒙古牛群体的染色体研究

分析了１５头（１０♂５♀）蒙古牛的核型，Ｃ带、Ｇ带和Ａｇ－ＮＯＲｓ．结果发现，蒙古牛的核型，Ｃ带和Ｇ带与普通牛（Ｂｏｓｔａｕｒｕｓ）相同，其公平核型为６０，ｘｙ母牛为６０，ｘｘ．ｙ染色体为一小的中着丝粒染色体，认为蒙古牛为普通牛型黄牛．观察统计了５９６个细胞的Ａｇ－ＮＯＲｓ数目，每细胞平均Ａｇ—ＮＯＲｓ数为５．８１８±０．５１３，变化范围为３～１０个．     

山西白羊草染色体核型分析

选用山西白羊草(Bothriochloa ischaemum)种子为材料,采用根尖压片法进行细胞染色体核型分析,为白羊草的种质资源利用及育种提供细胞学依据。观察统计了30个完整的中期分裂相确定染色体数目,并选其中5个分裂相进行核型分析。结果表明,白羊草染色体数为2n=40,核型公式为K(2n)=4x=40=34m+6sm,其中中部着丝粒染色体(m)为17对,近中部着丝粒染色体(sm)为3对,核型类型为1B。核型不对称系数为58.32%。     

Multifactorial Analysis of the Follicular Environment is Predictive of Oocyte Morphology in Cattle

Numerous attempts have been recently made in the search for a reliable, fast and noninvasive assay for selection of oocytes suitable for in vitro embryo production. Potential markers have been described in the follicle such as follicular fluid (FF) or cumulus cells (CCs). However, the reported findings are contradictory, which may reflect the complexity of metabolism of the ovarian follicle. In the present experiment, a data set from individual follicles of known diameter was obtained: cumulus-oocyte complex (COC) morphology, fatty acid composition and glucose concentration in FF as well as apoptotic index in CCs. The obtained data was statistically analyzed either separately (univariate analysis) or simultaneously (multivariate analysis) to examine its predictive value in morphology assessment of bovine COCs. Although the univariate analysis yielded a complex relation system of the selected parameters, no clear outcome could be established. In multivariate analysis, the concentration of the four fatty acids (C16:0, C16:1, C18:1cis9, C22:5n3) and Δ⁹-desaturase (16) as well as elongase activities were selected as covariates. This allowed prediction of the morphology of a COC with an accuracy of 72%, which is the most interesting finding of the experiment. The present study indicates that the multifactorial model comprising of selected parameters related to the follicle appeared more effective in predicting the morphology of a bovine COC, which may improve the effectiveness of in vitro production systems.     

紫花苜蓿离体组织染色体加倍技术及在育种中的应用

通过与传统的染色体加倍技术相比较,概述了用秋水仙素诱发离体组织染色体加倍的优点和具体的染色体加倍技术,探讨了秋水仙素诱发形成多倍体的细胞学机制,多倍体的鉴定方法,最后简述了染色体加倍技术在紫花苜蓿育种中的应用。     

Expression and Activation of Mitogen-activated Protein Kinases in Matured Porcine Oocytes under Thermal Stress

In this study, we determined the expression and activation of p38 MAPK in matured porcine oocytes subjected to heat shock (HS). When MII oocytes were heated, only the phosphorylated p38 levels relative to the total p38 levels decreased (P < 0.01) after HS, but no clear relationship with HS treatments was observed in the ERK, JNK and p90^rsk expressions of matured oocytes. To confirm p38 activation in matured oocytes, immunocytochemical staining was performed to localize its expression and distribution in the ooplasm, and the results were largely consistent with previous Western blot analyses. Moreover, when matured oocytes were co-cultured with a P38 MAPK inhibitor, SB203580, for 4 h at 41.5 C, the activation of its immediate downstream substrate MAPKAPK-2 was not inhibited within any of the treatment groups. It appears that the MAPKAPK2 levels increased only under prolonged culture (HS4h and C4h) compared with the control group. In conclusion, p38 activity in porcine oocytes was decreased after exposure to HS and prolonged culture. These alterations of p38 and activation of MAPKAPK2 may be associated with porcine oocyte viability under HS conditions, and a potential cross-talk between p38 MAPK and other signaling cascades may exist, which warrants additional investigation.     

牧草染色体核型和带型分析研究进展

植物染色体的核型和带型分析对物种基因图谱的构建、亲缘关系的确定、种质资源的保存、进化过程的探索、植物学分类及育种等方面的研究具有重要作用.分析了植物染色体核型和带型研究的异同点,并从取材、预处理、固定、解离、制片及染色等方面系统综述了牧草核型和带型分析的研究进展.核型和带型分析方法需要进一步的规范和标准化,在种质资源评价、种子劣变生理等方面将成为牧草核型与带型分析研究的重点.     

A Trial to Cryopreserve Immature Medaka (Oryzias latipes) Oocytes after Enhancing Their Permeability by Exogenous Expression of Aquaporin 3

Fish oocytes have not been cryopreserved successfully, probably because it is difficult to prevent intracellular ice from forming. Previously, we have shown in medaka that immature oocytes are more suitable for cryopreservation than mature oocytes or embryos, in terms of permeability. We have also shown in immature medaka oocytes that the exogenous expression of aquaporin 3 (AQP3), a water/cryoprotectant channel, promotes the movement of water and cryoprotectants through the plasma membrane. In the present study, we attempted to cryopreserve immature medaka oocytes expressing AQP3. We first examined effects of hypertonic stress and the chemical toxicity of cryoprotectants on the survival of the AQP3-expressing oocytes. Exposure to hypertonic solutions containing sucrose decreased the survival of oocytes, but the expression of AQP3 did not affect sensitivity to hypertonic stress. Also, AQP3 expression did not markedly increase sensitivity to the toxicity of cryoprotectants. Of the four cryoprotectants tested, propylene glycol was the least toxic. Using a propylene glycol-based solution, therefore, we tried to cryopreserve immature oocytes by vitrification. During cooling with liquid nitrogen, all intact oocytes became opaque, but many AQP3-expressing oocytes remained transparent. This indicates that the expression of AQP3 is effective in preventing intracellular ice from forming during cooling. During warming, however, all the AQP3-expressing oocytes became opaque, indicating that intracellular ice formed. Therefore, the dehydration and permeation by propylene glycol were still insufficient. Further studies are necessary to realize the cryopreservation of fish oocytes.     

Hematoxylin Staining Reveals a Decrease in Nucleolar Diameter of Pig Oocytes Before Germinal Vesicle Breakdown

During oocyte growth, the morphology of the nucleolus changes into a compact and homogenous structure. The compact nucleoli in full-grown oocytes are not stained by aceto-orcein staining or immunofluorescence staining. In this study, we developed a hematoxylin staining method for pig oocytes in whole-mount preparations to visualize the nucleoli. Nucleoli of growing and full-grown oocytes were stained blue with hematoxylin. Using this staining method, the changes in the oocyte nucleolus during maturation were examined. The nucleolar diameter gradually decreased in maturing oocytes (10.7 ± 0.1 μm to 9.0 ± 0.7 μm, P<0.05) before germinal vesicle breakdown (GVBD). The results suggest that the nucleolar volume of oocytes decreases before GVBD.     

Evaluation of Zona Pellucida Function for Sperm Penetration During In Vitro Fertilization in Pigs

In porcine oocytes, the function of the zona pellucida (ZP) with regard to sperm penetration or prevention of polyspermy is not well understood. In the present study, we investigated the effects of the ZP on sperm penetration during in vitro fertilization (IVF). We collected in vitro-matured oocytes with a first polar body (ZP+ oocytes). Some of them were freed from the ZP (ZP− oocytes) by two treatments (pronase and mechanical pipetting), and the effects of these treatments on sperm penetration parameters (sperm penetration rate and numbers of penetrated sperm per oocyte) were evaluated. There was no evident difference in the parameters between the two groups. Secondly, we compared the sperm penetration parameters of ZP+ and ZP− oocytes using frozen-thawed epididymal spermatozoa from four boars. Sperm penetration into ZP+ oocytes was found to be accelerated relative to ZP− oocytes. Thirdly, we evaluated the sperm penetration of ZP+ and ZP− oocytes at 1−10 h after IVF (3 h gamete co-incubation). The proportions of oocytes penetrated by sperm increased significantly with time in both groups; however, the number of penetrated sperm per oocyte did not increase in ZP− oocytes. Finally, we performed IVF using ZP− oocytes divided into control (3 h) and prolonged gamete co-incubation (5 h) groups. Greater numbers of sperm penetrated in the 5 h group than in the control group. These results suggest that the ZP and oolemma are not competent factors for prevention of polyspermy in our present porcine IVF system. However, it appears that ZP removal is one of the possibilities for reducing polyspermic penetration in vitro in pigs.     

徐淮白山羊染色体核型研究1

采用外周血淋巴细胞培养及染色体分带技术分析了徐淮白山羊的染色体核型、C-带。结果表明，徐淮白山羊二倍体染色体数为2N=60，常染色体和X染色体均为端部着丝粒染色体．X染色体的大小介于1号和2号染色体之间，Y染色体最小．为中部着丝粒染色体，公羊核型为60．XY．母羊为60．XX。大部分常染色体和性染色体着丝粒部位显示阳性C带。     

黄芪和甘草染色体数目及核型分析

运用植物根尖压片法，对黄芪和甘草两种豆科植物的染色体数目、核型作了研究。结果表明，黄芪的染色体基数X＝8，甘草的染色体基数X＝7。两个种都是二倍体，核型分别为2B和1A，核型公式分别为：2n=2x=16=14m 2sm和2n=2x=14=14m。