体外受精和孤雌活化过程中小鼠胚胎细胞骨架的动态变化

Dynamic changes of microtubule in parthenogenetic and in vitro fertilized preimplantation embryos in mouse

为研究微管在体外受精与孤雌活化过程中的动态变化，本实验比较了体外受精胚胎、SrCl2激活的孤雌胚胎和体内受精的原核期胚胎在体外发育的情况，采用免疫荧光化学与激光共聚焦显微术检测卵母细胞孤雌活化过程中及体外受精后微管及核的动态变化，以分析微管在减数分裂过程中的作用及其对早期发育的影响。结果显示，体内受精胚胎的发育率显著高于体外受精和孤雌激活胚胎体外发育率（P〈0．05），而体外受精与孤雌激活胚胎在各阶段发育率差异均不显著。在体外受精中，精子入卵，激活卵母细胞，减数分裂恢复，纺锤丝牵拉赤道板上致密排列的母源染色体向纺锤体两侧迁移：后期将染色体拉向两极；末期时，微管分布于两组已去凝集的母源染色体之间，卵母细胞排出第二极体（the second polarbody，Pb2），解聚的母源染色体形成雌原核。同时，在受精后5—8h精子染色质发生去浓缩与再浓缩，形成雄原核。在原核形成的同时，胞质星体在雌、雄原核的周围重组形成长的微管，负责雌、雄原核的迁移靠近。孤雌活化过程中，卵母细胞恢复减数分裂，姐妹染色单体分离，被拉向两极，经细胞松弛素B处理后，活化4—6h，卵周隙中未见Pb2，而在胞质中出现两个混合的单倍体原核，之间由微管相连接，负责两个单倍体原核的迁移靠近。与体外受精相比较，孤雌活化时卵母细胞更容易被激活，减数分裂期间微管的发育早且更完善。

In this study we detected dynamic changes and function of β-tubulin, a subtype of microtubule, during the first cleavage period in mouse parthenogenetic and in vitro fertilized embryos. Firstly, we compared the developmental potential of in vitro fertilized, parthenogenetic, and in vivo fertilized embryos in culture. Then, the dynamic changes of β-tuhulin and nucleus in parthenogenetic and in vitro fertilized preimplantation embryos were detected by immunofluorescence and confocal microscopy to analyze the role of microtubules in meiotic division and embryonic development. The results indicated that the development rate of in vivo fertilized embryos was significantly higher than that of in vitro fertilized or parthenogenetic embryos （P〈0.05）. However, there was no significant difference in developmental potential between in vitro fertilized and parthenogenetic embryos. During in vitro fertilization, oocyte was activated when sperm entered it. Oocyte resumed the second meiotic division. Condensed maternal chromosomes aligning at the equator of the spindle were pulled to the spindle poles by kinetochore microtubules in anaphase. Furthermore, in telophase, there were microtubules between the two sets of decondensed maternal chromosomes. One set formed the second polar body （Pb2）, which was extruded to the perivitelline space. The other set formed female pronucleus. Meanwhile, 5-8 h after fertilization, sperm chromatin condensed and decondensed to form male pronucleus. Microtubule composed mesosome and cytaster remodeling around male and female pronuclei to form long microtubules, which pull the pronuclei to get close. During 4-6 h parthenogenetic activation, SrCl2 activated oocytes to resume meiosis. As a consequence, sister chromatids were pulled to spindle poles. Cytochalasin B, which was applied in the medium, inhibited the extrusion of Pb2. Two haploid pronuclei in the cytoplasm were connected by microtubules. Compared with that in in vitro fertilization, oocyte is easier to be activated in parthenogenetic activation. Chemical activation is more efficient than sperm penetration in in vitro fertilization as indicated by earlier and better remodeling of the microtubules.