Van Wissen B, Bomsel-Helmreich O, Debey P, Eisenberg C, Vautier D, Pennehouat G
U187 Hôpital A. Béclère, Clamart, France.
Hum Reprod. 1991 Jul;6(6):879-84. doi: 10.1093/oxfordjournals.humrep.a137444.
Some human oocytes cultured together with spermatozoa for in-vitro fertilization (IVF) do not subsequently divide. The arrest of the fertilization process at different moments during development may provide information about the cause of fertilization failure. Oocytes which subsequently divide are transferred 48 h after insemination; when oocytes do not divide, ageing processes can be observed. Therefore these oocytes are interesting material in which to observe both fertilization and ageing. Our study concerns 72 undivided human oocytes 0, 48 or 72 h post-insemination. DNA of the oocyte and spermatozoa was visualized by the DNA fluorescent dye Hoechst 33342. Living oocytes were observed in toto by fluorescence and bright field microscopy which allowed nuclear and pronuclear membranes to be discerned. Oocytes were subsequently fixed and sectioned for bright field microscopy. Both techniques allowed parallel observations. Oocytes at various stages of fertilization are described: sperm penetration in both mature and immature oocytes, decondensation of sperm-heads, premature condensation of male chromatin, polyspermy and pronucleus formation. Typical ageing processes such as the centripetal migration of the metaphase II chromosomes, the formation of a restitution nucleus and the lagging of chromosomes within a metaphase spindle are observed. DNA fluorescence appears to be a quick, easy and valuable means to analyse fertilization and its failure.
一些与精子一起培养用于体外受精（IVF）的人类卵母细胞随后并不会分裂。受精过程在发育的不同时刻停滞可能会提供有关受精失败原因的信息。随后分裂的卵母细胞在授精后48小时被转移；当卵母细胞不分裂时，可以观察到老化过程。因此，这些卵母细胞是观察受精和老化的有趣材料。我们的研究涉及72个授精后0、48或72小时未分裂的人类卵母细胞。卵母细胞和精子的DNA用DNA荧光染料Hoechst 33342进行可视化。通过荧光和明场显微镜对活卵母细胞进行整体观察，这使得能够辨别核膜和原核膜。随后将卵母细胞固定并切片用于明场显微镜观察。这两种技术都能进行平行观察。描述了处于受精不同阶段的卵母细胞：精子穿透成熟和未成熟卵母细胞、精子头部解聚、雄性染色质过早凝聚、多精受精和原核形成。观察到典型的老化过程，如中期II染色体向心迁移、重建核的形成以及染色体在中期纺锤体内的滞后。DNA荧光似乎是分析受精及其失败的一种快速、简便且有价值的方法。
