Involvement of histone H2B monoubiquitination in the regulation of mouse preimplantation development

Histone H2B monoubiquitination (H2Bub1) plays an important role in developmental regulation in various vertebrate species. However, the role of H2Bub1 in mammalian preimplantation development remains unclear. In the present study, we examined the role of H2Bub1 in the regulation of mouse preimplantation development. Based on immunocytochemical analysis using an anti-H2Bub1 antibody, no H2Bub1 signal was detected in the metaphase chromosomes of unfertilized oocytes or the pronuclei of early 1-cell stage embryos, but a weak signal was observed in late 1-cell stage embryos. The signal increased after cleavage into the 2-cell stage, and thereafter a strong signal was observed until the blastocyst stage. To assess the significance of H2Bub1 in the regulation of preimplantation development, RNF20 (an H2B-specific ubiquitin E3 ligase) was knocked down using small interfering RNA (siRNAs). In embryos treated with siRNA, the levels of Rnf20 mRNA and H2Bub1 decreased at the 4-cell and morula stages. Although these embryos developed normally until the morula stage, only one-third developed into the blastocyst stage. These results suggested that H2Bub1 is involved in the regulation of preimplantation development.     

Stored of Hsp72/Hsp73 in Germinal Vesicle-stage Mouse Oocytes

Heat-shock proteins (hsps) Hsp72 and Hsp73 are the stored maternal proteins found in mouse oocytes. Both hsps appear in mouse oocytes at germinal vesicle (GV) and metaphase II (M-II)-stages as previously demonstrated by immunoblotting analysis. In this report, we further determined the presences of Hsp72/Hsp73 proteins in mouse embryos at stages of 2-pronucleus, arrested 1-cell, 2-cell, arrested 2-cell, 4-cell, arrested 4-cell, 8-cell to morula and blastocyst. Except for the blastocyst stage, the Hsp72/Hsp73 proteins were detectable in most embryo stages. The concentration of Hsp72/Hsp73 in GV-stage oocytes was higher than that in M-II-stage oocytes, and in any stages of embryos before implantation. A dramatical increase in Hsp72/Hsp73 expression was found at the 2-cell stage. Together with these findings, we speculated that hsps accumulated or stored earlier in the GV-stage mouse oocytes to protect the oocytes against environmental influences acting on ovary, and hsps may be required for zygotic gene activation and provided a protective effect against apoptosis.     

Inheritance of histone H3 methylation in reprogramming of somatic nuclei following nuclear transfer

Successful cloning requires reprogramming of epigenetic information of the somatic nucleus to an embryonic state. However, the molecular mechanisms regarding epigenetic reprogramming of the somatic chromatin are unclear. Herein, we transferred NIH3T3 cell nuclei into enucleated mouse oocytes and evaluated the histone H3 dimethyl-lysine 4 (H3K4me2) dynamics by immunocytochemistry. A low level of H3K4me2 in the somatic chromatin was maintained in pseudo-pronuclei. Unlike in vitro fertilized (IVF) embryos, the methylation level of nuclear transfer (NT) embryos was significantly increased at the 8-cell stage. NT embryos showed lower H3K4me2 intensity than IVF embryos at the 2-cell stage, which is when the mouse embryonic genome is activated. Moreover, the H3K4me2 signal was weak in the recloned embryos derived from single blastomeres of the NT embryos, whereas it was intense in those from IVF embryos. Two imprinted genes, U2afbp-rs and Xist, were abnormally transcribed in cloned embryos compared with IVF embryos, and this was partly correlated to the H3K4me2 level. Our results suggest that abnormal reprogramming of epigenetic markers such as histone acetylation and methylation may lead to dysregualtion of gene expression in cloned embryos.     

牛体外受精早期胚胎发育基因差异表达的研究

应用mRNA差异显示技术,对牛卵母细胞、体外培养的牛早期8细胞期和囊胚期胚胎的基因表达进行了研究。选取4条mRNA表达量存在差异的基因条带进行测序和同源性分析,并利用RT-PCR技术粗略检测其在卵母细胞、8细胞和囊胚中的表达水平。结果表明:4个基因分别与核糖体蛋白S4,Y连接1(RPS4Y1)、末端脱氧核苷酰转移酶作用因子2(DNTTIP2)、剪接和多聚腺苷酸化特异因子3(CPSF3)和转录因子AP-2γ(TFAP2C)高度同源。RPS4Y1、DNTTIP2、CPSF3和TFAP2C在牛卵母细胞和早期胚胎发育过程中mRNA表达量存在时间性差异,可能与其参与不同的生理活动有关。     

Cleavage-embryo genes and transposable elements are regulated by histone variant H2A.X

During mammalian preimplantation development, stimulation of zygotic genome activation (ZGA) and transposable elements (TEs) shapes totipotency profiling. A rare mouse embryonic stem cells (mESCs) subpopulation is capable of transiently entering a state resembling 2-cell stage embryos, with subtypes of TEs expressed and ZGA genes transiently activated. In this study, we found that deletion of H2A.X in mESCs led to a significant upregulation of ZGA genes and misregulated TEs. ChIP-seq analysis indicated a direct association of H2A.X at the Dux locus for silencing the Dux gene and its downstream ZGA genes in mESCs. We also demonstrated that histone variant H2A.X is highly enriched in human cleavage embryos when ZGA genes and TEs are active. Therefore, we propose that H2A.X plays an important role in regulating ZGA genes and TEs to establish totipotency.     

山羊早期胚胎发育的初步研究

本实验以黑龙江地方山羊为材料,经FSH超数排卵后,在不同时间屠宰母山羊,并冲洗输卵管及子宫,获取新鲜卵及各发育时间的胚胎。实验中发现山羊的排卵时间为发情开始后约30小时。受精卵的第一次卵裂的发生在排卵24小时以后。2细胞、4细胞、8细胞、16细胞、桑椹及胚泡期胚胎所处的时间分别为排卵后约32～42、48～52、62、72、96小时以及7～8天。16细胞期以前的胚胎移行于输卵管中,桑椹胚及胚泡则移行于子宫中。在每一时间从每只羊中所收集的胚胎基本上处于几个相邻的发育时期。在桑椹胚的动物极有明显的突起,这可能是内细胞群已开始形成的标志。到胚泡期时,胚胎体积增大,透明带变薄。正在孵化或已经孵化的胚泡中,内细胞群外端无滋养层细胞包围。     

The effect of ovary storage and in vitro maturation on mRNA levels in bovine oocytes; a possible impact of maternal ATP1A1 on blastocyst development in slaughterhouse-derived oocytes

Since BSE testing of slaughtered cattle is obligatory in Japan, storage of ovaries at 15-20 C overnight in phosphate buffered saline has become a routine protocol in in vitro production (IVP) of cattle embryos. Ovary storage is known to reduce developmental competence of oocytes; however, its effects on oocyte gene expression have not been clarified yet. This study compared oocytes collected from stored slaughterhouse-derived ovaries with those collected by Ovum Pick-Up (OPU) in terms of the expression of 20 selected genes to determine if ovary storage affects cellular processes at the molecular level. Expression of mRNA in oocytes was assayed before and after in vitro maturation (IVM) by real-time quantitative PCR. Maternal mRNA levels of genes were investigated in 2-cell stage embryos obtained from slaughterhouse oocytes to assess their roles for blastocyst formation. In immature OPU oocytes, genes related to metabolism (GAPDH), transporters (GLUT8, ATP1A1) and stress resistance protein (HSP70) showed significantly higher expression compared with oocytes derived from stored ovaries. During IVM, the expression of GDF9, GLUT8, CTNNB1 and PMSB1 was significantly decreased irrespective of oocyte source. Two-cell stage embryos cleaving at 22-25 h after in vitro fertilization (IVF) showed a significantly higher blastocyst formation rate and ATP1A1 gene expression level compared with those cleaving at 27-30 h after IVF. Our results reveal that storage of ovaries alters mRNA levels in oocytes. Correlation of Na/K ATPase ATP1A1 expression in IVP embryos at the 2-cell and 8-cell stages with their developmental ability to the blastocyst stage may suggest the importance of maternal mRNA of this gene during blastulation in embryos derived from slaughterhouse oocytes.     

Open-pulled straw (OPS) vitrification of in vitro fertilised mouse embryos at various stages

The present study was designed to investigate the cryotolerance of in vitro fertilised (IVF) mouse embryos at various preimplantation developmental stages. IVF mouse embryos were vitrified by the open-pulled straw (OPS) method. After warming, embryos were morphologically evaluated and assessed by their development to blastocysts, hatched blastocysts or term. The results showed that a high proportion (93.3-100.0%) of vitrified embryos at all developmental stages were morphologically normal after recovery. The developmental rate of vitrified 1-cell embryos to blastocyst (40.0%) or hatched blastocyst (32.7%) or term (9.3%) was significantly lower than that from other stages (P < 0.05). Vitrified embryos from 2-cell to early blastocyst stage showed similar blastocyst (71.8-89.5%) and hatched blastocyst rates (61.1-69.6%) and could develop to term without a significant loss of survival compared with those of fresh embryos (P > 0.05). Vitrified 2-cell embryos showed the highest survival rate in vivo (50.6%, 88/174), compared with that from other stages (9.3-30.5%, P < 0.05). The data demonstrate that the OPS method is suitable for the cryopreservation of IVF mouse embryos from 2-cell stage to early blastocyst stage without a significant loss of survival. Embryos at the 2-cell stage had the best tolerance for cryopreservation in the present study.     

Developmental ability of somatic cell nuclear transferred embryos aggregated at the 8-cell stage or 16- to 32-cell stage in cattle

Aggregation of somatic cell nuclear transfer (SCNT) embryos in mice is reported to improve full-term development. In the present study, we attempted to improve the development of SCNT embryos by aggregation in cattle. In Experiment 1, to examine the effect of the timing of aggregation on in vitro development of cumulus-cell NT embryos, we aggregated two or three SCNT embryos (2X or 3X embryos) at the 1-cell, 8-cell and 16- to 32-cell stages. Irrespective of the timing of aggregation, 3X embryos developed to the blastocyst stage at a high rate. However, aggregation did not improve the total blastocyst formation rate of the embryos used. The cell numbers of 3X embryos aggregated at the 1-cell stage and 2X embryos tended to be higher than that of single NT embryos (1X embryos). Furthermore, a significant increase in cell number was observed in 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. In Experiment 2, we used fibroblast cells as nuclear donors and examined in vitro development of 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. As a result, 3X embryos had high blastocyst formation rates and higher cell numbers than 1X embryos, which was consistent with the results of Experiment 1. In Experiment 3, we examined the full-term developmental ability of 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. After transfer of fibroblast-derived NT embryos into recipient animals, a significantly higher pregnancy rate was obtained on Day 60 in 3X embryos than in 1X embryos. Two embryos aggregated at 8-cell stage and one embryo aggregated at the 16- to 32-cell stage developed to term, while no pregnancies derived from 1X embryos that lasted to Day 60. However, two of the cloned calves were stillborn. These results suggest that aggregation of the 8-cell stage or 16- to 32-cell stage SCNT embryos may improve the pregnancy rate, but that it cannot reduce the high incidence of fetal loss and stillbirth, which is often observed in bovine SCNT.