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Production of normal mice from spermatozoa denatured with high alkali treatment before ICSI

¹ Laboratory for Genomic Reprogramming, RIKEN, Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan² Department of Bioscience, Graduate School of Science and Technology, Kwansei Gakuin University, Sanda 669-1337, Japan³ Department of Medical Science, Graduate School of Medicine, Kyoto University, Kyoto 606-8502, Japan

Correspondence should be addressed to C Li; Email: lichong{at}cdb.riken.jp

In mammals, ICSI is now a very important tool for both assisted reproductive technology and studying the mechanisms of fertilization. In the latter experiments, it is important to use spermatozoa that have lost their oocyte activation capacity but still retain their developmental potential. In this study, we used high-concentration NaOH to remove oocyte activation potential from spermatozoa, and examined whether normal offspring could be generated from these spermatozoa after ICSI. The spermatozoa were treated with different concentrations of NaOH (1–100 mM) for 1 h and then neutralized with equal amounts of same concentration of HCl. In 10 mM NaOH-treated spermatozoa, the cell membrane was broken and most of them failed to activate oocytes after their injection into the oocytes. However, these spermatozoa did not show strong damage, and after artificial activation with SrCl₂, all of the zygotes were judged as normal by immunostaining to check the methylation status of histone H3 lysine 9, low chromosome damage by karyotype assay and staining with DNA double-strand breaks marker, H2AX. Moreover, after transferring those embryos into recipient females, 106 (36.7%) live and healthy offspring were delivered, which is similar to the rate in the fresh control group. By contrast, spermatozoa treated with lower NaOH concentrations retained their oocyte activation capacity and those treated with higher concentrations lost their developmental potential. This suggests that 10 mM NaOH for 1 h is the best treatment to completely destroy the cell membrane and activation capacity of spermatozoa without injuring their developmental potential.

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