School of Public and Health, Guilin Medical University, Guilin, 541004, China.
Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, China.
J Assist Reprod Genet. 2020 Dec;37(12):3057-3067. doi: 10.1007/s10815-020-01975-4. Epub 2020 Oct 21.
This study aimed to determine the effects of drilling and thinning treatment of laser-assisted hatching on the expression and methylation of imprinted gene IGF2/H19 in embryos and offspring.
The prehatching blastocysts with treatment of drilling or thinning, or control prehatching blastocysts, were transplanted in surrogate uteri. The DNA methylation of IGF2/H19 imprinting control region (ICR) and the expression of IGF2 and H19 were respectively evaluated using bisulfite conversion-mediated sequencing and real-time PCR.
The drilling group showed a significant increase in the development rate of hatched blastocysts in comparison with the control and thinning group. DNA methylation level of IGF2/H19 ICR of hatched blastocysts in the thinning group was 27.33% in comparison with the 38.67% and 36% observed in the control and drilling group. The thinning treatment increased the DNA methylation level of IGF2/H19 ICR in the placenta in comparison with the control and drilling group. The drilling and thinning treatment decreased the expression level of H19 mRNA in prehatching and hatched blastocysts as well as placenta, while a significant increase in the expression level of H19 mRNA of offspring was observed in the thinning group. The thinning treatment increased the expression level of IGF2 mRNA of prehatching blastocysts and offspring and a significant decrease in placenta, while the drilling treatment resulted in a significant increase in the expression level of IGF2 mRNA of hatched blastocysts and placenta.
These observations suggested that drilling used for hatching of in vitro cultured mouse blastocysts may improve the production of offspring.
本研究旨在探讨激光辅助孵化钻孔及削薄处理对胚胎及子代印迹基因 IGF2/H19 表达和甲基化的影响。
对打孔或削薄处理的孵化前囊胚或对照孵化前囊胚进行移植。采用亚硫酸氢盐转化介导测序和实时 PCR 分别评估 IGF2/H19 印迹控制区（ICR）的 DNA 甲基化和 IGF2 和 H19 的表达。
与对照组和削薄组相比，打孔组孵化囊胚的发育率显著提高。与对照组和打孔组相比，削薄组孵化囊胚 IGF2/H19 ICR 的 DNA 甲基化水平为 27.33%，而对照组和打孔组分别为 38.67%和 36%。与对照组和打孔组相比，削薄处理增加了胎盘 IGF2/H19 ICR 的 DNA 甲基化水平。打孔和削薄处理降低了预孵化和孵化囊胚以及胎盘的 H19 mRNA 表达水平，而削薄组子代的 H19 mRNA 表达水平显著增加。削薄处理增加了预孵化囊胚和子代的 IGF2 mRNA 表达水平，并显著降低了胎盘的 IGF2 mRNA 表达水平，而打孔处理则导致孵化囊胚和胎盘的 IGF2 mRNA 表达水平显著增加。
这些观察结果表明，用于体外培养的囊胚孵化的钻孔处理可能会提高后代的产量。
