Pan Xiaoyan, Kong Delong, Liu Limei, Gao Fei, Zhang Xueming, Tang Bo, Li Ziyi
Jilin Provincial Key Laboratory of Animal Embryo Engineering, the Center for Animal Embryo Engineering of Jilin Province, College of Veterinary Medicine, Jilin University, Changchun, Jilin 130062, China; Department of Histology and Embryology, Jilin Medical College, Jilin, Jilin 132013, China.
Cell Biol Int. 2014 Nov;38(11):1280-90. doi: 10.1002/cbin.10319. Epub 2014 Jun 6.
We have investigated the mechanism for embryo development block in vitro and to improve the development rate of golden hamster embryos in vitro. Intracytoplasmic sperm injection (ICSI) technique was used to produce golden hamster ICSI embryos. The changes in the histone acetylation and the expression of histone deacetylase and related genes were analyzed by immunocytochemical staining and real-time PCR both in golden hamster in vivo embryos and in ICSI embryos. Aged oocytes significantly increased the oocyte spontaneous activation rate. In vitro cultured ICSI embryos suffered from severe development block in M199TE medium. Expression of histone deacetylase 1 (HDAC1) was significantly decreased in the nuclei of the arrested ICSI 2-cell embryos, and its nuclear and cytoplasmic expression pattern was also markedly altered. The acetylation level of H4K5, however, was not significantly changed between golden hamster in vivo embryos and ICSI embryos. HSPA1A and MYC, the marker genes for zygotic genome activation (ZGA), were transcriptionally decreased in arrested ICSI 2-cell embryos. Transcription of HDAC1 was also downregulated in these embryos, whereas the mRNA expression of the proapoptotic gene, BAX, was not changed. These results indicate that the golden hamster ICSI embryo development block during ZGA is associated with decreased nuclear expression and altered expression of HDAC1. HSPA1A, MYC, and HDAC1 mRNA levels, which decrease, resulting in ZGA failure.
我们研究了体外胚胎发育阻滞的机制,以提高金黄仓鼠胚胎的体外发育率。采用胞浆内单精子注射(ICSI)技术制备金黄仓鼠ICSI胚胎。通过免疫细胞化学染色和实时PCR分析了金黄仓鼠体内胚胎和ICSI胚胎中组蛋白乙酰化的变化以及组蛋白去乙酰化酶和相关基因的表达。老化卵母细胞显著提高了卵母细胞的自发激活率。体外培养的ICSI胚胎在M199TE培养基中遭受严重的发育阻滞。在停滞的ICSI 2细胞胚胎的细胞核中,组蛋白去乙酰化酶1(HDAC1)的表达显著降低,其核内和胞浆内的表达模式也明显改变。然而,金黄仓鼠体内胚胎和ICSI胚胎之间H4K5的乙酰化水平没有显著变化。合子基因组激活(ZGA)的标记基因HSPA1A和MYC在停滞的ICSI 2细胞胚胎中转录水平降低。这些胚胎中HDAC1的转录也下调,而促凋亡基因BAX的mRNA表达没有变化。这些结果表明,金黄仓鼠ICSI胚胎在ZGA期间的发育阻滞与HDAC1核表达降低和表达改变有关。HSPA1A、MYC和HDAC1的mRNA水平降低,导致ZGA失败。