昆明鼠卵母细胞的孤雌发育及对其干扰核移植试验的预防措施

本文着重研究了在昆明鼠细胞核移植过程中，卵母细胞孤雌发育的激活原因、形成过程，并提出了一些防止其干扰细胞核移植的措施。     

不同浓度CO2对牛卵母细胞体外成熟和受精的影响

对牛卵巢卵泡内卵母细胞在不同浓度的CO2培养箱内体外成熟、体外受精后的卵裂率及胚胎体外发育进行了研究。结果显示,含5%小牛血清(CS)的TCM-199内的卵母细胞在2%CO2条件下培养20h后,达到第二次减数分裂期(MⅡ期)的卵子数明显高于在5%CO2条件下培养的卵母细胞数(P<0.05)。两种浓度的CO2条件下培养的牛卵母细胞体外受精后的卵裂率无明显差异,2%CO2浓度下培养的卵母细胞的囊胚发育率高于5%CO2浓度下培养的卵母细胞。     

Nuclear transfer in loach (Paramisgurnus dabryanus Sauvage) by cell-to-cell electrofusion

To study nuclear transfer in the loach (Paramisgurnus dabryanus Sauvage), blastula and gastrula cells were fused with UV-inactivated oocytes by cell-to-cell electrofusion. To facilitate nuclear transfer, blastula and gastrula cells were cultured or incubated at 4 °C in different solutions. TC-199 medium supplemented with 20% calf serum was the best culture solution, and effectively retained the totipotence of blastula or gastrula cells for up to 10 days. It was found that gastrula cells incubated at 4 °C had the same totipotence as blastula cells. The optimal UV dosage for inactivation of the oocyte chromatin was 180–240 mJ cm⁻². Electrofusion was carried out in a cone-shaped fusion chamber, which permitted the recipient oocyte and the donor blastula cell to contact one another. The electrofusion procedure resulted in a 10% success rate of normal-appearing fish. Genetic analysis indicated that the nuclear material originated from the donor cell (blastomere) and the oocyte pronucleus did not take part in development.     

Cumulus Oophorus Expansion of Bovine Oocytes Cultured in vitro: A SEM and TEM Study

Bovine cumulus-oocyte complexes (COCs) were aspirated from antral follicles (3–6 mm in diameter). COCs with a compact multilayered cumulus investment were cultured for 9, 13 and 24 hours, respectively, and processed for scanning or transmission electron microscopy after different periods of culture including a 0 hour control group. In all 0 hour control COCs initial formation of cumulus cell projections, blebs and microvilli were observed. The start of cumulus-oocyte disconnection in cattle was observed after 9 hours of in vitro culture. These connections were almost completely discrupted after 13 hours, when a continuous production of extracellular matrix was observed. The full expansion of corona radiata cells was not observed even after 24 hours. Some cumulus oophorus cells were bound together with junctions of the zonula adherens type after 24 hours when extracellular matrix was found only in deeper layers. The full expansion of corona radiata cells was not the prerequisite for disconnection of cumulus-oocyte complex. Inhalt: Kumulus oophorus Expansion von in vitro kultivierten Rinderoozyten: Eine SEM und TEM Untersuchung Kumulus-Oozyten-Komplexe (COC) von Rindern wurden aus antralen Follikeln aspiriert (Durchmesser 3–6 mm). COC's mit kompakten mehrschichtigen Kumulus wurden für 9, 13 und 24 Stunden kultiviert und für die Scanner- oder Transmissionselektronenmikroskopie vorbereitet. Verschiedene Kulturzeiten incl. einer 0-Stundengruppe als Kontrolle wurden untersucht. In allen Kontroll COC's wurden beginnende Formationen yon Kumuluszellprojektionen, Bläschen und Mikrovilli beobachtet. Nach 9-stündiger in vitro Kultur begann die Lösung zwischen Kumulus und Oozyte. Die Verbindungen waren nach 13 Stunden vollständig unterbrochen, wobei eine kontinuierliche Produktion extrazellulärer Matrix beobachtet wurde. Eine vollständige Expansion der Corona radiata Zellen wurde auch nicht nach 24 Stunden beobachtet. Nach 24 Stunden waren einige Cumulus Oophorus Zellen über Verbindungen des Zonula adherens Typs verknüpft, wobei extrazelluläre Matrix nur in den tieferen Zellschichten gefunden wurde. Eine vollständige Kumulusexpansion ist keine Voraussetzung für die Auflösung des Kumulus-Oozyten-Komplexes.     

昆明鼠卵母细胞的去核及其去核率的观察

本文研究昆明鼠卵母细胞的去核方法，并通过对去核卵母细胞的固定染色，显示其中期Ⅱ染色体，由此观察统计其确定的去核率。     

细胞松弛素B浓度对小鼠卵母细胞去核效果的影响

目的　探讨细胞松弛素 B浓度对小鼠卵母细胞去核效果的影响。方法　常规超排 KM小鼠获卵母细胞 ,将有明显第一极体的卵母细胞放入含有 5× 10 - 3、1× 10 - 2 、2× 10 - 2 g/ L CB的成熟培养液中孵育 15 min,采用盲吸法去核 ,去核后的卵母细胞放入含 5× 10 - 3g/ L Hoechst33342的成熟培养液中染色 15 min,在荧光显微镜下检查去核的效率。结果　 CB组去核率可达 88.2 % ,而对照组去核成功率仅达 2 9.7% ,两者的去核效果统计学上有显著差异 ;同时 CB的浓度过高会影响去核的效果 ,使卵母细胞的破溃率升高。结论　 CB有利于去核 ,同时卵胞膜可以借助磷脂双层的游动性而很快得到修复 ;高浓度的 CB对卵胞膜作用过于强烈 ,细胞骨架严重破坏 ,磷脂双层结构的弹性和粘性减弱 ,修复能力降低 ,影响胚胎的构建。     

Meiotic competence of mouse oocytes reconstructed by replacing the germinal vesicle with a male pronucleus and somatic nucleus

The present study examines the contribution of the nucleus to meiotic competence in mouse oocytes that were reconstructed using nuclear transfer. Three types of reconstructed oocytes were produced: MP‐GV, by transplanting the male pronucleus (MP) into germinal vesicle (GV) stage oocytes; 3T3‐GV, by transplanting the nucleus of a National Institute of Health (NIH) 3T3 cell into a GV stage oocyte; and 3T3‐MII, by transplanting the nucleus of an NIH 3T3 cell into a metaphase II (MII) stage oocyte. The fusion rates differed, but not significantly, in the MP‐GV, 3T3‐GV, and 3T3‐MII groups (77, 63, 56%, respectively). Then, meiotic competence was compared in MP‐GV, 3T3‐GV and non‐manipulated GV stage oocytes as a control. Nuclear envelope breakdown occurred in all the reconstructed oocytes, as well as the control ones. The percentage of first polar body extrusion differed between the MP‐GV (100%), 3T3‐GV (72%), and control (67%) groups. DNA staining with Hoechst 33342 revealed that in the MP‐GV‐group oocytes that had reached MII stage, the chromosomes were condensed and aligned in a regular array similar to the normal metaphase plate. By contrast, in 3T3‐GV group oocytes, the condensed chromosomes were irregularly scattered in the cytoplasm. These results suggest that the donor nucleus affects meiotic competence in reconstructed oocytes.     

BPA不影响卵母细胞减数分裂相关基因Dazl的甲基化

为了探讨环境雌激素BPA对小鼠卵母细胞减数分裂相关基因Dazl甲基化的影响,本研究通过给孕鼠饮用含有BPA的水方式使胎鼠在发育过程中接触BPA,利用重亚硫酸盐测序法,分析了胎鼠生殖嵴卵母细胞不同发育时期Dazl甲基化水平的变化。结果显示:Dazl在减数分裂期间处于低甲基化水平,无论对照组或处理组均低于10%,说明Dazl的低甲基化对维持减数分裂的正常进行有重要作用;对照组与处理组的甲基化水平相当,差异不显著,说明本研究的BPA浓度不影响卵母细胞Dazl的甲基化水平。