Expression of N-myc in teratocarcinoma stem cells and mouse embryos

The N-myc gene, which is distantly related to the proto-oncogene c-myc, was first detected as an amplified sequence in human neuroblastoma cell lines and tumours. It has since been revealed that there is up to a 300-fold amplification of N-myc DNA in almost 50% of advanced metastatic human neuroblastomas, whereas amplification is not detected in less advanced tumours that have a better prognosis (ref.3 and M.S., unpublished data). Although expression of N-myc is detectable in all neuroblastoma cell lines and tumours examined, its level is greatly enhanced when the N-myc gene is amplified. Recently, it has been shown that on co-transfection with the c-Ha-ras (EJ) gene, N-myc can induce the malignant transformation of rat embryo fibroblasts. Taken together, these data imply a function for N-myc in the development and/or progression of human neuroblastomas. Surveys indicate that N-myc also may be amplified and/or expressed in two other types of human tumours and cell lines derived from them: retinoblastomas and small cell lung cancers. Here, we report that N-myc is expressed at high levels in mouse and human teratocarcinoma stem cells, thus identifying another tumour cell type that expresses the N-myc gene. In addition, we found that N-myc is abundantly expressed in mouse embryos at mid-gestation and that its expression appears to decrease as the embryo approaches term. In the adult mouse, N-myc is expressed at an approximately fivefold lower level in the brain than in teratocarcinoma stem cells and embryos, and at even lower levels in the adult testis and kidney. Our data represent the first demonstration of expression of the N-myc gene in normal cells, and suggest that N-myc may be involved in mammalian embryogenesis.     

Aberrant DNA methylation patterns in cultured mouse embryos

Mouse early embryos undergo genome-wide demethylation and remethylation events during pre-implantation development. Abnormal methylation reprogramming is thought to be associated with development arrest. Using immunofluorescence staining with an antibody against 5-methylcytosine (MeC), we examined the genome methylation patterns of mouse embryos cultured in vitro. The results did not show the difference in staining patterns between development-blocked two-cell embryos that cultured in vitro and the two-cell embryos that were freshly collected from the donor mice. But in vitro-arrested morulae displayed a strong positive staining when compared to the morulae freshly collected from the donor mice. At the blastocyst stage, although most embryos showed the expected methylation patterns, with highly stained inner cell mass (ICM) and weekly stained trophectoderm (TE), a proportion of embryos were dimly stained in both ICM and TE. These results indicated that the methylation profile of the embryos could be changed by culturing in vitro when the embryos were in the transition from morulae to blastocyst.     

小鼠早期胚胎的全胚原位杂交技术

目的:探索一种适合于早期胚胎基因表达型检测的全胚原位杂交技术.方法:收集小鼠囊胚、固定;制备VASP、IL1R2基因特异性地高辛标记的cRNA探针;对囊胚进行杂交前处理、全胚原位杂交、抗体处理、显色以及显色后处理与镜检.结果:VASP、IL1R2特异性表达在囊胚的内细胞团细胞和滋养层细胞中.结论:小鼠早期胚胎全胚原位杂交技术适用于早期胚胎基因时空表达型的检测.     

Derivation of haploid embryonic stem cells from mouse embryos

Most animals are diploid, but haploid-only and male-haploid (such as honeybee and ant) species have been described. The diploid genomes of complex organisms limit genetic approaches in biomedical model species such as mice. To overcome this problem, experimental induction of haploidy has been used in fish. Haploid development in zebrafish has been applied for genetic screening. Recently, haploid pluripotent cell lines from medaka fish (Oryzias latipes) have also been established. In contrast, haploidy seems less compatible with development in mammals. Although haploid cells have been observed in egg cylinder stage parthenogenetic mouse embryos, most cells in surviving embryos become diploid. Here we describe haploid mouse embryonic stem cells and show their application in forward genetic screening.     

Effect of monoclonal antibody to bull sperm sp18 on murine fertilization and embryos development

Western blot analysis revealed that one IgG1 monoclonal antibody (mAb) to sp18 family membrane proteins (Mr. 14, 16 and 18 ku) of bovine sperm reacted faintly with protein bands of 14, 18, 22, 30 and 60 ku (reducing) in samples of mouse sperm. The mAb also reacted to protein of egg lysozyme. Using a laser confocal microscope, indirect immunofluorescence (IIP) showed that the sp18 antigens were present in the posterior head of murine sperm. In murine in vitro fertilization (IVF) and embryo development trails, a total of 426 oocytes from C57BL/6 and F1 hybrid strain (CD1 × C57BL/6 cross) of 12 female mice were used in 3 independent trails. After preincubating capacitated sperm with 182 μg/mL of sp18 mAb in the modified TYH IVF medium for 15-20 min, cumulus-oocyte-complexes were introduced. The fertilization rate in sp18 mAb groups was 77.1 %, which was not significantly (P > 0.05) different from the nonspecific mouse IgG (79.2%) and non-IgG (80.3 %) control groups. Fertilized oocytes had been continuously cultured in modifed CZB medium. 100%, 100% and 97.9% of 1-cell embryos developed to 2-cell stage in sp18 mAb, nonspecific mouse IgG and non-IgG group 30 h after the start of fertilization, respectively. In the nonspecific mouse IgG and non-IgG groups, 64.1 % and 64.3% of embryos developed to the 4-cell stage, respectively, but all developing eggs in sp18 mAb groups arrested development in vitro at 2-cell stage. After zonae of 2-cell blocked embryos were enzy-matically removed with 0.5% pronase, detection of sp18 antigens by IIF indicated that the fluorescence scattered on two embryonic cells. For embryos fertilized in vivo and co-cultured with 182 μg/mL sp18 mAb, the numbers of 1-cell embryos reaching the 2-cell and 4-cell stage were 95. 2% and 70. 5%, which were not significantly (P>0.05) different from the control group (92.9% and 77.9%). These results indicate that the sp18 antigens on posterior head of mouse sperm were incorporated into the egg plasma membrane during fertilization, and played an active role in development of murine preimplanta-tion embryo.     

Calcium-releasing activity induced by nuclei of mouse fertilized early embryos

At fertilization,repectitive transient rises of intracellular calcium concentration occur in all mammals studied so far .It has been shown that calcium rises could be induced when mouse fertilized 1-,2-cell nuclei were trans-planted into unfertilized eggs and that the reconstituted embryo could be activated .Howerver,whecther the capability of inducing calcium rises occurs in all stages of mammalian embryos remains unknown ,In this study ,by using the nuclear transplantation technique and measurement of intracellular calcium rises in living cells,we showed that only the nuclei from mouse fertilized 1-cell and 2-cell embryos ,neither the nuclei from 4-,8-cell and ethanol activated parthe-nogenetic embryos nor 2 or 3 nuclei of electrofused 4-cell stage syncytium ,have calcium -releasing activity when they were transferred into unfertilized mature oocytes,Our results indicate that the calcium-releasing activity in nuclei of 1-,2-cell embryos is produced during fertilization and exists at the special stage of fertilized early embryos.These sug-gested that the capacity of inducting calcium release activity in fertilized early embryos is important for normal embryonic development.