Abnormal distribution of chromosomes in the first division of nuclear transferred mouse embryos

The majority of somatic cell nuclear transferred (SCNT) embryos die before or after implantation. Many studies have focused on morphological remodeling of the donor nucleus and its associated cytoskeletal structures in the early events of nuclear transfer. However, little is known about the 2-cell stage of SCNT embryos after the first division. In this study, we compared the morphological status of chromosomal division during the 1-cell stage to the 2-cell stage in SCNT embryos with that in intracytoplasmic sperm injection (ICSI) embryos. The microtubules and cytoplasmic asters, which are related to chromatin segregation, disappeared at the pronuclear stage, although formation of the first mitotic spindle was normal in both the SCNT and ICSI embryos. However, nuclear fragmentation was observed in 30% of the 2-cell SCNT embryos and 12% of the 2-cell ICSI embryos. Nuclear fragmentation was present in both blastomeres of these embryos. No apoptotic DNA fragmentation was observed in TdT-mediated dUTP-biotin Nick End Labeling (TUNEL) assays for either the SCNT or ICSI embryos. In both the SCNT and ICSI embryos, the distribution of chromosomes in the first mitotic spindle was disturbed during the process of division from the 1-cell stage to the 2-cell stage. These results suggest that loss of SCNT embryos just before or after implantation may be due to an abnormal chromosome distribution at the 2-cell stage.     

斜纹夜蛾核型多角体病毒Ⅱ型分离株ORF63基因的序列特征及表达与启动子活性分析

斜纹夜蛾核型多角体病毒Ⅱ型(SpltMNPVⅡ)分离株是繁殖率和毒力极强的新型病毒株,ORF63是该病毒分离株的一个功能未知基因。从SpltMNPVⅡ分离株基因组中克隆了ORF63。序列分析表明,该基因读码框为1 071 bp,编码356个氨基酸,蛋白质分子质量为41.3 kD;起始密码子ATG上游存在一个早期和晚期启动子基序,编码蛋白序列含有CNX、MutH等多种结构域。启动子活性和转录时相分析表明ORF63是一个早、晚期表达的基因,在病毒感染4 h和18 h时有2个转录峰,24 h以后转录水平略有下降,但总体趋于稳定。构建该基因的原核表达载体pET-28a-ORF63,并转化大肠杆菌BL21(DE3),表达并纯化融合蛋白后制备多克隆抗体,Western blot检测制备的多克隆抗体特异性较好,效价可达1∶3 200以上。由以上结果推测,SpltMNPVⅡ分离株的ORF63基因是一个早期和晚期均有表达的病毒基因,可能与SpltMNPVⅡ感染宿主细胞后自身DNA的复制有关,参与早期芽生病毒(BV)的发生和晚期包涵体衍生病毒(ODV)的成熟2个过程。     

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.     

Mouse in vivo-derived late 2-cell embryos have higher developmental competence after high osmolality vitrification and −80°C preservation than IVF or ICSI embryos

Mammalian embryos are most commonly cryopreserved in liquid nitrogen; however, liquid nitrogen is not available in special environments, such as the International Space Station (ISS), and vitrified embryos must be stored at −80°C. Recently, the high osmolarity vitrification (HOV) method was developed to cryopreserve mouse 2-cell stage embryos at −80°C; however, the appropriate embryo is currently unknown. In this study, we compared the vitrification resistance of in vivo-derived, in vitro fertilization (IVF)-derived, and intracytoplasmic sperm injection (ICSI)-derived mouse 2-cell embryos against cryopreservation at −80°C. The ICSI embryos had lower survival rates after warming and significantly lower developmental rates than the in vivo and IVF embryos. Further, IVF embryos had a lower survival rate after warming, but a similar rate to the in vivo embryos to full-term development. This result was confirmed by simultaneous vitrification of in vivo and IVF embryos in the same cryotube using identifiable green fluorescent protein-expressing embryos. We also evaluated the collection timing of the in vivo embryos from the oviduct and found that late 2-cell embryos had higher survival and developmental rates to full-term than early 2-cell embryos. Some early 2-cell embryos remained in the S-phase, whereas most late 2-cell embryos were in the G2-phase, which may have affected the tolerance to embryo vitrification. In conclusion, when embryos must be cryopreserved under restricted conditions, such as the ISS, in vivo fertilized embryos collected at the late 2-cell stage without long culture should be employed.     

In vitro development of IVF-derived bovine embryos following cytoplasmic microinjection for the episomal expression of the IGF2 gene

Important genomic imprinting changes usually occur following the in vitro production (IVP) of bovine embryos, especially in the imprinting pattern of components of the IGF system. This study aimed to evaluate the effects of a transient episomal overexpression of the IGF2 gene in bovine IVP embryos following embryo cytoplasmic microinjection (CMI) at the 1-cell stage on embryo survival, early and late developmental kinetics and morphological quality up to Day 7 of development. Selected cumulus–oocyte complexes (COCs) were matured and fertilized in vitro and subsequently segregated into six experimental groups: non-CMI control group and five CMI groups at increasing doses (0, 10, 20, 40 and 80 ng/μl) of a GFP vector built for the episomal expression of bovine IGF2. Zygote CMI was effective in delivering the expression vector into the ooplasm, irrespective of the groups, with 58% of positive GFP fluorescence in Day 7 blastocysts. Considering developmental rates and late embryo kinetics, the 10-ng/μl CMI vector dose promoted a lower blastocyst rate (10.4%), but for blastocysts at more advanced stages of development (93.0% blastocysts and expanded blastocysts), and higher number of cells (116.0 ± 3.0) than non-CMI controls (23.3%, 75.0% and 75.0 ± 6.8 were obtained, respectively). In conclusion, CMI at the 1-cell stage did not compromise subsequent in vitro development of surviving embryos, with the 10-ng/μl group demonstrating a possible growth-promoting effect of the IGF2 gene on embryo development, from the 1-cell to the blastocyst stage.     

H3S10 Phosphorylation Marks Constitutive Heterochromatin During Interphase in Early Mouse Embryos Until the 4-Cell Stage

Phosphorylation of histone H3 at Ser10 (H3S10P) has been linked to a variety of cellular processes, such as chromosome condensation and gene activation/silencing. Remarkably, in mammalian somatic cells, H3S10P initiates in the pericentromeric heterochromatin during the late G2 phase, and phosphorylation spreads throughout the chromosomes arms in prophase, being maintained until the onset of anaphase when it gets dephosphorylated. Considerable studies have been carried out about H3S10P in different organisms; however, there is little information about this histone modification in mammalian embryos. We hypothesized that this epigenetic modification could also be a marker of pericentromeric heterochromatin in preimplantation embryos. We therefore followed the H3S10P distribution pattern in the G1/S and G2 phases through the entire preimplantation development in in vivo mouse embryos. We paid special attention to its localization relative to another pericentromeric heterochromatin marker, HP1β and performed immunoFISH using specific pericentromeric heterochromatin probes. Our results indicate that H3S10P presents a remarkable distribution pattern in preimplantation mouse embryos until the 4-cell stage and is a better marker of pericentromeric heterochromatin than HP1β. After the 8-cell stage, H3S10P kinetic is more similar to the somatic one, initiating during G2 in chromocenters and disappearing upon telophase. Based on these findings, we believe that H3S10P is a good marker of pericentromeric heterochromatin, especially in the late 1- and 2-cell stages as it labels both parental genomes and that it can be used to further investigate epigenetic regulation and heterochromatin mechanisms in early preimplantation embryos.     

Molecular dynamics of heterochromatin protein 1beta, HP1beta, during mouse preimplantation development

To elucidate the molecular dynamics of HP1beta in mouse preimplantation embryos, we examined the localization, dynamics, and mobility of HP1beta in the (pro)nucleus by live cell imaging. Time-lapse observation revealed that the chromatin association of HP1beta is regulated in a cell cycle-dependent manner. HP1beta was localized in the interphase nucleus and was dynamically dissociated from the nucleus during the metaphase stage. The HP1beta assembly and clustered heterochromatin structure were both found in the nuclei of 2-cell and later-stage embryos. Moreover, fluorescent recovery after photobleaching analysis implied that HP1beta is more freely mobile in the pronucleus of the 1-cell embryo than in the 4-cell nucleus. These results suggest that the chromatin configuration may be regulated by the stability and mobility of chromatin-associated proteins including HP1beta during early embryonic stages.     

Demands for carbohydrates as major energy substrates depend on the preimplantation developmental stage in pig embryos: Differential use of fructose by parthenogenetic diploids before and after the 4-cell stage in the pig

The embryo culture technique has been improving, but the detailed demands for energy substrates such as glucose, fructose, pyruvate and lactate of preimplantation embryos are still unclear. In the present study, the demands of pig preimplantation embryos at each different developmental stage were investigated by use of parthenogenetic diploids as a model of pig preimplantation embryos. Pig parthenogenetic diploids showed different use of glucose and fructose before and after the 4-cell stage. Although glucose supported the development of pig embryos throughout the preimplantation stages and even maintained the expansion and hatching of blastocysts, it suppressed development to the blastocyst stage when glucose coexisted with pyruvate and lactate from 4 h after activation, but not after 48 h (early 4-cell stage). Since ketohexokinase that metabolizes fructose was not expressed in 2-cell and 4-cell diploids, a medium that included only fructose as a major energy substrate did not support early cleavage of pig diploids beyond the 4-cell stage, and almost no diploids developed to the morula stage just as in a medium without carbohydrates. These results may explain the different suppressive effects on pig preimplantation development between glucose and fructose when pyruvate and lactate were present in a medium. In addition, 4-cell diploids that had been cultured in a medium with pyruvate and lactate developed to the expanded blastocyst stage without any carbohydrates as a major energy substrate. These results show that the demands for carbohydrates are different depending on the developmental stage in pig preimplantation embryos.     

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.     

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.     

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.     

Interspecies nuclear transfer embryos reconstructed from cat somatic cells and bovine ooplasm

This study was conducted to investigate the developmental capacity of domestic cat-bovine reconstructed embryos via interspecies somatic cell nuclear transfer (iSCNT) and to observe the mitochondrial DNA (mtDNA) content of the iSCNT embryos. The iSCNT embryos were generated using mixed-breed domestic cat fibroblasts as donor cells and enucleated bovine oocytes as the recipient cytoplasm. When the developmental capacities of iSCNT embryos and parthenogenic bovine embryos were compared, there was no difference (P>0.05) in the rates of cleavage and development to the 8-cell stage (86.6 vs. 84.0% and 32.2 vs. 36.2%, respectively). However, in contrast to development of parthenogenic embryos to the morula and blastocyst stages, no iSCNT embryos (0/202) developed beyond the 8-cell stage. For mtDNA analysis, iSCNT embryos at the 1-cell, 2-cell, 4-cell and 8-cell stages were randomly selected. Both cat and bovine mtDNA quantification analysis were performed using quantitative PCR. The levels of both cat and bovine mtDNA in cat-bovine iSCNT embryos varied at each stage of development. The cat mtDNA concentration in the iSCNT embryos was stable from the 1-cell to 8-cell stages. The bovine mtDNA in the iSCNT embryos at the 8-cell stage was significantly lower than that at the 4-cell stage (P<0.05). No difference in the proportions of cat mtDNA in the iSCNT embryos was found in any of the observed developmental stages (1- through 8-cell stages). In conclusion, bovine cytoplasm supports domestic cat nucleus development through the 8-cell stage. The mtDNA genotype of domestic cat-bovine iSCNT embryos illustrates persistence of heteroplasmy, and the reduction in mtDNA content might reflect a developmental block at the 8-cell stage.     

Timing and uniformity of embryonic gene activation affect subsequent pre-implantation development of cloned bovine embryos

In the present study, we examined the timing of onset, intensity, and mosaicism of embryonic gene expression in bovine nuclear transfer (NT) embryos. The relationship between gene expression and early embryonic development was also examined. To monitor the gene expression of NT embryos, we produced NT embryos with bovine transfected fibroblasts carrying a firefly luciferase gene under the control of a chicken beta-actin promoter, an expression system that has previously been shown to be representative of embryonic gene expression in mice. Photon count imaging showed that luciferase luminescence began in NT embryos with fibroblasts 48 hours post fusion (hpf) and reached a plateau at the 4- to 8-cell stage at 60 hpf. Only 4- to 8-cell NT embryos luminescent by 60 hpf developed to the blastocyst stage. At 60 hpf, strongly luminescent embryos developed to the blastocyst stage at a higher rate (P<0.05) than embryos with weak or absent luminescence. However, embryos with mosaic luminescence developed at a much lower rate (P<0.05) than those with whole-embryo luminescence, even if the embryos exhibited strong luminescence. Our results indicate that precise and uniform embryonic gene expression at the 4- to 8-cell stage at 60 hpf may be closely related to development of bovine NT embryos to the blastocyst stage.     

Meiotic arrest with roscovitine and sexual maturity improve competence of mouse oocytes by regulating expression of competence-related genes

We have studied the mechanisms by which meiotic arrest maintenance (MAM) with roscovitine, female sexual maturity, and the surrounded nucleoli (SN) chromatin configuration improve the competence of mouse oocytes by observing the expression of oocyte competence-related genes in non-surrounded nucleoli (NSN) and SN oocytes from prepubertal and adult mice following maturation with or without MAM. The results demonstrated that MAM with roscovitine significantly improved the developmental potential of adult SN and prepubertal NSN oocytes, but had no effect on that of prepubertal SN oocytes. Without MAM, while 40% of the 2-cell embryos derived from prepubertal SN oocytes developed into 4-cell embryos, none of the 2-cell embryos derived from prepubertal NSN oocytes did, and while 42% of the 4-cell embryos derived from adult SN oocytes developed into blastocysts, only 1% of the 4-cell embryos derived from prepubertal SN oocytes developed into blastocysts. Furthermore, MAM with roscovitine, SN configuration, and female sexual maturity significantly increased the mRNA levels of competence-beneficial genes and decreased those of competence-detrimental genes. In conclusion, our results suggest that MAM with roscovitine, SN chromatin configuration, and female sexual maturity improve oocyte competence by regulating the expression of competence-related genes, suggesting that Oct4, Stella, Mater, Zar1, Mapk8, and Bcl2 are oocyte competence-beneficial genes, whereas Foxj2, Ship1, and Bax are competence-detrimental genes.     

Expression of the Orexigenic Peptide Ghrelin and the Type 1a Growth Hormone Secretagogue Receptor in Sheep Oocytes and Pre-implantation Embryos Produced In Vitro

Recent studies have implicated the peripheral actions of ghrelin in reproductive tissues. Here, we provide evidence that both ghrelin and its receptor GHSR-1a (the type 1a growth hormone secretagogue receptor) are expressed at both mRNA and protein levels in sheep oocytes and pre-implantation embryos produced in vitro . Real-time RT-PCR experiments confirmed that ghrelin mRNA levels varied depending on developmental stage, with the highest expression in metaphase II (MII) oocytes, higher expression at the 2-cell stage, and minimal expression in germinal vesicle (GV) oocytes, 4- and 8-cell stages, and in the blastocyst. The levels of GHSR-1a mRNA decreased from GV to MII, increased immediately at the 2-cell stage and then remained stable until the blastocyst stage. Ghrelin protein was detected mainly in the cytoplasm close to the plasma membrane in both inner cell mass and trophectoderm cells, while GHSR-1a protein was most abundant in the plasma membrane. In conclusion, the presence of the ghrelin signalling system within the sheep oocytes and pre-implantation embryos opens up the possibility of a potential regulatory role of this novel molecule in reproductive function.