Study On Round Spermatid Injection In The Mouse For Assisted Reproductive Technology

Abstract

Round Spermatid Injection (ROSI) is widely considered as an alternative infertility treatment for men who do not be able to produce mature sperm due to some reasons related abnormal spermatogenesis (Tanaka, et al., 2018). However, the successful rate of blastocyst formation which leads to significantly low birth rate from such procedure is being recorded with unexplained underlying mechanisms that might be due to abnormal epigenetic modifications based on previous researches on inbred and hybrid mouse strains (Kishigami S, et al., 2004; Ohta H, et al., 2009; Ogonuki N, et al., 2011). Nevertheless, study of Round Spermatid Injection (ROSI) on ICR strain- an outbred mouse species, has not been conducted yet despite its strong characteristics for oocyte manipulation. Therefore, in this project, the objective is to establish an effective ROSI system on this mouse strain, as well as to investigate the preimplantation development of embryos injected with testicular round spermatids from the zygote to the blastocyst stage in comparison with Intracytoplasmic Sperm Injection (ICSI) embryos - a control group. The results indicated that ROSI embryos have similar ability of developing into blastocysts as ICSI embryos, although the significant lower embryonic cell number in DAPI stained blastocysts count. Interestingly, the injection of only round spermatid nucleus resulted in the correlation between pronuclear formation to the late-stage development in ICSI group was just slightly higher than the ROSI-generated oocytes, with 93% and nearly 83% in turn. Moreover, DAPI staining pictures of embryos from both groups showed that round spermatid - derived oocytes possessed quite similar morphology and nuclei quality as those in ICSI embryos at various stages in terms of preimplantation developmental competence. On the other hand, the examination of Histone H3K9 Methylation expression pointed out the significantly-remaining methylation status of male pronucleus of ROSI zygotes at 6 hour after activation of injected oocytes which directly contradicted to the demethylated status in ICSI male pronucleus at the same time interval. Accordingly, to sum up, in this study, we successfully established the effective ROSI procedure on ICR mouse strain that generated similar embryonic developmental competence with other mouse strains. Additionally, we discovered the remain methylation of ROSI male pronucleus at the 6-hour zygote stage that possibly affected the abnormal paternal reprogramming at the preimplantation development of embryos.