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

Abstract

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 NaOHtreated 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 SrCl 2 , 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, gH2AX. 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.

TRPV3 Channels Mediate Strontium-Induced Mouse-Egg Activation

“…Sr 2+ has been widely used to induce parthenogenesis in rodent eggs (Kono et al, 1996; Li et al, 2009; Tomashov-Matar et al, 2005). The channel that mediates Sr 2+ influx has not been reported in any species, and whether Sr 2+ and Ca 2+ permeate the same channel(s) in mouse eggs is unknown.…”

Characterization of the ICSI‐mediated gene transfer method in the production of transgenic pigs

“…The connection between the sperm cell membrane and the exogenous DNA is considered to be important in determining the transfer efficiency of this DNA by ICSI‐MGT (Perry et al, 1999). There are also reports that use of sperm in which the cell membrane has been removed by alkaline treatment increases the efficiency of production of transgenic mice by ICSI‐MGT (Li et al, 2009). We therefore suggest that the extent of damage to the sperm cell membrane greatly influences the frequency of mosaic embryos obtained by ICSI‐MGT.…”

Development of novel intracytoplasmic sperm injection and somatic cell nuclear transfer techniques for animal reproduction

“…Recently, as mentioned above (Li et al . 2009), we developed a sperm demembranation method using a NaOH treatment.…”

“…Therefore, we conducted tests that involved the removal of the cellular membrane before adding the cryoprotectant. When sperm were treated with 10 mmol NaOH for 1 h, we succeeded in perfectly removing the cellular membrane without damaging the nucleus, although the spermatozoa died immediately by this treatment (Li et al . 2009).…”