Ogonuki Narumi, Inoue Kimiko, Miki Hiromi, Mochida Keiji, Hatori Masanori, Okada Hironori, Takeiri Shuji, Shimozawa Nobuhiro, Nagashima Hiroshi, Sankai Tadashi, Ogura Atsuo
RIKEN Bioresource Center, Tsukuba, Ibaraki, Japan.
Mol Reprod Dev. 2005 Nov;72(3):411-7. doi: 10.1002/mrd.20363.
Microinsemination is the technique of delivering male germ cells directly into oocytes. The efficiency of fertilization after microinsemination and subsequent embryo development may vary with the animal species and male germ cells used. The present study was undertaken to observe the in vitro and in vivo developmental ability of rabbit embryos following microinsemination with male germ cells at different stages. First, we assessed their oocyte-activating capacity by injecting them into mouse and rabbit oocytes. The majority of mouse oocytes were activated irrespective of the type of rabbit male germ cell injected (61-77%), whereas rabbit oocytes were activated differently according to the type of male germ cells (89%, 75%, and 29% were activated by spermatozoa, elongated spermatids, and round spermatids, respectively; P < 0.05). After 120 hr in culture, 66%, 45%, and 13%, respectively, of these activated rabbit oocytes (pronuclear eggs) developed into blastocysts (P < 0.05). Additional electric pulse stimulation of round spermatid-injected oocytes increased the blastocyst rate to 43%. After 24 hr in culture, some four to eight cell embryos were transferred into the oviducts of pseudopregnant females. Normal pups were born from spermatozoa and elongated spermatids, but not from round spermatids. Karyotypic analysis at the morula/blastocyst stage revealed that the majority of round spermatid-derived embryos had abnormal ploidy (8 out of 12 embryos). Our study indicates that rabbit male germ cells acquire the ability to activate oocytes and to support subsequent embryo development as they undergo spermiogenesis. As these differential developmental patterns are similar to those reported for humans in vitro and in vivo, rabbits may provide an alternative small animal model for studying the biological nature and molecular basis of human microinsemination techniques, especially those using immature male germ cells.
显微授精是将雄性生殖细胞直接注入卵母细胞的技术。显微授精后的受精效率以及随后的胚胎发育可能因动物物种和所使用的雄性生殖细胞而异。本研究旨在观察用不同阶段的雄性生殖细胞进行显微授精后兔胚胎的体外和体内发育能力。首先，我们通过将它们注入小鼠和兔的卵母细胞来评估其卵母细胞激活能力。无论注入的兔雄性生殖细胞类型如何，大多数小鼠卵母细胞都被激活（61%-77%），而兔卵母细胞根据雄性生殖细胞类型的不同而有不同的激活情况（分别有89%、75%和29%的卵母细胞被精子、延长型精子细胞和圆形精子细胞激活；P<0.05）。培养120小时后，这些被激活的兔卵母细胞（原核卵）分别有66%、45%和13%发育成囊胚（P<0.05）。对注入圆形精子细胞的卵母细胞进行额外的电脉冲刺激可使囊胚率提高到43%。培养24小时后，将一些4至8细胞胚胎转移到假孕雌性的输卵管中。正常幼崽由精子和延长型精子细胞发育而来，但圆形精子细胞则不能。桑椹胚/囊胚阶段的核型分析显示，大多数圆形精子细胞来源的胚胎具有异常的倍性（12个胚胎中有8个）。我们的研究表明，兔雄性生殖细胞在经历精子形成过程中获得了激活卵母细胞并支持随后胚胎发育的能力。由于这些不同的发育模式与人类体外和体内报道的模式相似，兔子可能为研究人类显微授精技术的生物学性质和分子基础，特别是那些使用未成熟雄性生殖细胞的技术，提供一种替代的小动物模型。
