Ogonuki Narumi, Inoue Kimiko, Ogura Atsuo
RIKEN BioResource Center, Ibaraki, Japan.
J Reprod Dev. 2011 Sep;57(4):534-8. doi: 10.1262/jrd.11-008m. Epub 2011 Mar 26.
For fertilization using round spermatid injection (ROSI) in mice, oocytes need to be artificially preactivated because of the lack of oocyte-activating capacity in round spermatids of this species. However, when round spermatids were frozen-thawed before microinjection, 11-71% of injected oocytes developed into 2-cell embryos without any artificial activation. After being transferred into recipient females, 5-27% of these embryos reached term. At least some of the injected oocytes showed intracellular Ca(2+) oscillations, which normally occur after fertilization by mature spermatozoa. Thus, these round spermatids could transmit a sperm-borne oocyte-activating factor, which might have been released from spermatozoa and elongated spermatids in the same suspension by freezing and thawing. This possibility was further supported by activation of intact oocytes following transplantation of the pronuclei from ROSI-generated embryos. Thus, one-step ROSI can be achieved in mice simply by injecting frozen-thawed round spermatids into intact oocytes. Clearly, there is a need for careful interpretation of microinjection experiments when assessing the oocyte-activating capacity of spermatogenic cells, especially when they are derived from frozen-thawed stocks.
在小鼠中使用圆形精子细胞注射（ROSI）进行受精时，由于该物种圆形精子细胞缺乏卵母细胞激活能力，卵母细胞需要进行人工预激活。然而，当圆形精子细胞在显微注射前进行冻融处理时，11%-71%的注射卵母细胞在没有任何人工激活的情况下发育成2-细胞胚胎。将这些胚胎移植到受体雌性体内后，5%-27%的胚胎发育至足月。至少一些注射的卵母细胞显示出细胞内Ca(2+)振荡，这通常在成熟精子受精后发生。因此，这些圆形精子细胞可能传递了一种精子携带的卵母细胞激活因子，该因子可能通过冻融从同一悬浮液中的精子和延长型精子细胞中释放出来。将ROSI产生的胚胎的原核移植后完整卵母细胞的激活进一步支持了这种可能性。因此，在小鼠中，只需将冻融的圆形精子细胞注射到完整卵母细胞中，即可实现一步ROSI。显然，在评估生精细胞的卵母细胞激活能力时，尤其是当它们来自冻融储备时，需要对显微注射实验进行仔细解读。
