Tremoleda Jordi L, Van Haeften Theo, Stout Tom A E, Colenbrander Ben, Bevers Mart M
Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 12, 3584 CM Utrecht, The Netherlands.
Biol Reprod. 2003 Jul;69(1):186-94. doi: 10.1095/biolreprod.102.012823. Epub 2003 Mar 19.
Intracytoplasmic sperm injection (ICSI) is the method of choice for fertilizing horse oocytes in vitro. Nevertheless, for reasons that are not yet clear, embryo development rates are low. The aims of this study were to examine cytoskeletal and chromatin reorganization in horse oocytes fertilized by ICSI or activated parthenogenetically. Additional oocytes were injected with a sperm labeled with a mitochondrion-specific vital dye to help identify the contribution of the sperm to zygotic structures, in particular the centrosome. Oocytes were fixed at set intervals after sperm injection and examined by confocal laser scanning microscopy. In unfertilized oocytes, microtubules were present only in the metaphase-arrested second meiotic spindle and the first polar body. After sperm injection, an aster of microtubules formed adjacent to the sperm head and subsequently enlarged such that at the time of pronucleus migration and apposition it filled the entire cytoplasm. During syngamy, the microtubule matrix reorganized to form a mitotic spindle on which the chromatin of both parents aligned. Finally, after nuclear and cellular cleavage were complete, the microtubule asters dispersed into the interphase daughter cells. Sham injection induced parthenogenetic activation of 76% of oocytes, marked by the formation of multiple cytoplasmic microtubular foci that later developed into a dense microtubule network surrounding the female pronucleus. The finding that a parthenote alone can produce a microtubule aster, whereas the aster invariably forms at the base of the sperm head during normal fertilization, indicates that both gametes contribute to the formation of the zygotic centrosome in the horse. Finally, 25% of sperm-injected oocytes failed to complete fertilization, mostly due to absence of oocyte activation (65%), which was often accompanied by failure of sperm decondensation. In conclusion, this study demonstrated that union of the parental genomes in horse zygotes is accompanied by a series of integrated cytoskeleton-mediated events, failure of which results in developmental arrest.
胞浆内单精子注射（ICSI）是体外使马卵母细胞受精的首选方法。然而，由于尚不清楚的原因，胚胎发育率较低。本研究的目的是检查通过ICSI受精或孤雌激活的马卵母细胞中的细胞骨架和染色质重组。另外，向卵母细胞中注射用线粒体特异性活性染料标记的精子，以帮助确定精子对合子结构的贡献，特别是对中心体的贡献。在精子注射后按设定的时间间隔固定卵母细胞，并通过共聚焦激光扫描显微镜检查。在未受精的卵母细胞中，微管仅存在于中期停滞的第二次减数分裂纺锤体和第一极体中。精子注射后，微管星状体在精子头部附近形成，随后扩大，使得在原核迁移和并置时它充满整个细胞质。在配子融合期间，微管基质重新组织形成有丝分裂纺锤体，双亲的染色质在其上排列。最后，在核分裂和细胞分裂完成后，微管星状体分散到间期子细胞中。假注射诱导76%的卵母细胞发生孤雌激活，其特征是形成多个细胞质微管灶，这些微管灶后来发展成围绕雌原核的致密微管网络。单独的孤雌胚能够产生微管星状体，而在正常受精过程中微管星状体总是在精子头部基部形成，这一发现表明，在马中两个配子都对合子中心体的形成有贡献。最后，25%的注射精子的卵母细胞未能完成受精，主要是由于卵母细胞未激活（65%），这通常伴随着精子去浓缩失败。总之，本研究表明，马合子中亲代基因组的结合伴随着一系列由细胞骨架介导的整合事件，这些事件的失败会导致发育停滞。
