Research Of Mouse Haploid Round Spermatid Microinjection

Some patients with non-obstructive azoospermia(NOA)can get their own offspring with paternal genetic material by the microinjection with haploid round spermatids into oocytes(round spermatid microinjection,ROSI).The haploid spermatids can be obtained from semen or by testicular biopsy.Although some ROSI-generated babies had been successfully born,at present there are still many unresolved problems,such as poor development potential of ROSI embryos and dramatically lower birthrate than that of ICSI.Therefore,it is necessary to explore these problems and find out new ways to improve the efficiency of ROSI,which will make this assisted reproduction technology(ART)more widely used in human clinical.In recent years,some research reported that ART increased the risk of suffering from the imprinting defects related diseases,so the safety of ART became the focus of current study.Consequently,besides exploring how to improve the efficiency of ROSI,we aslo detected the methylation states of two typical imprinting genes,which was able to provide certain preliminary reference for evaluating the epigenetic risk of the ROSI technology.The content of this study includes two parts:(Ⅰ)First,mouse round spermatids were treated with germinal vesicle(GV)cytolysates and injected this round spermatid alone or co-injected with GV oocyte nucleolus into mature metaphase II oocytes,the subsequent embryonic development potential was analyzed.Combined with immunofluorescence technique,the quality of blastocysts was assessed by morphological characteristics and the number of Oct4 positive cells in inner cell mass.And the level of histone H3K9me3 in ROSI embryos was detected through the immune fluorescence intensity analysis.The results indicated that there was no significant difference between experimental groups and control group at the zygote to four-cell development stages.Blastocysts derived from oocytes which were injected with GV cytolysates treated-round spermatid alone or co-injected with nucleoli injection yielded 63.63% and 70.83% high quality embryos(Grade A),respectively;higher than these oocytes which were co-injected with lysis buffer-treated round spermatids and nucleoli(50.00%)or injected with the lysis buffer-treated round spermatids alone(44.44%).Furthermore,the proportion of live offspring resulting from oocytes which were co-injected with cytolysates treated-round spermatids and nucleoli or injected with cytolysates treated-round spermatids alone was higher than those were injected with lysis buffer treated-round spermaids(55.07%,50.82% vs.36.73%,P<0.05),but comparable with the ICSI group.In addition,the quantitative analysis results of fluorescence intensity showed that GV cytolysates treatment could significantly reduce the signal of H3K9me3 in ROSI-derived embryos,aproaching the levels of ICSI counterparts.Our results demonstrated that factors from the GV cytoplasm could improve round spermatid reprogramming,and significantly facilitate ROSI technology.While the injection of an extra nucleolus did not obviously improve reprogramming,its potential contribution to reprogramming could not be definitively excluded.(Ⅱ)In this part of the study,we investigated the effects of the histone deacetylase inhibitor,Scriptaid,on the development of mice ROSI-derived embryos.Our results showed that 250 nM scriptaid treated ROSI-derived embryos for 10 h after microinjection resulted in higher blastocysts formation rate than that of the untreated ROSI-derived embryos(59.14% vs.39.53%,P<0.05),and further improved live offspring birth rates(40.00% vs.20.59%,P<0.05).Furthermore,the Scriptaid treated ROSI-derived embryos increased several pluripotent genes(Oct4,Nanog and Sox2)expression,rendering the expression levels similar to those of the ICSI counterparts.And we aslo found that the ICRs of H19 and Snrpn genes appeared demethylation and mosaic-type methylation pattern in ROSI-derived blastocysts.However,Scriptaid treated embryos were prone to maintain the original DNA methylation states of imprinted genes at blastocysts stage,consistent with ICSI counterparts.The results showed that the appropriate Scriptaid treatment can enhance the potential preimplantation development of ROSI-derived embryos in vitro,and then improve the efficiency of ROSI.ROSI-derived embryo treated with Scriptaid had up-regulated some pluripotency genes and repaired the abnormal methylation states of imprinted genes,comparable with ICSI counterparts,which may suggest that Scriptaid treatment improved the reprogramming of embryos derived from ROSI.These data indicated that Scriptaid treatment could improve the full-term development of embryos generated from spermatids,and aslo serve as a warning of the potential epegenetic risks of ROSI in human.In order to accurately evaluate its safety,further comprehensive study is needed.In conclusion,for the first time we find that treatment with GV cytolysates or Scriptaid can improve the efficiency of ROSI in mouse model,which provides a new insight into the improvement of the efficiency and safety of ROSI in clinical attempts to resolve severe human male infertility.