Putative imprinted gene expression in uniparental bovine embryo models

Altered patterns of gene expression and the imprinted status of genes have a profound effect on cell physiology and can markedly alter embryonic and fetal development. Failure to maintain correct imprinting patterns can lead to abnormal growth and behavioural problems, or to early pregnancy loss. Recently, it has been reported that the Igf2R and Grb10 genes are biallelically expressed in sheep blastocysts, but monoallelically expressed at Day 21 of development. The present study investigated the imprinting status of 17 genes in in vivo, parthenogenetic and androgenetic bovine blastocysts in order to determine the prevalence of this unique phenomenon. Specifically, the putatively imprinted genes Ata3, Impact, L3Mbtl, Magel2, Mkrn3, Peg3, Snrpn, Ube3a and Zac1 were investigated for the first time in bovine in vitro fertilised embryos. Ata3 was the only gene not detected. The results of the present study revealed that all genes, except Xist, failed to display monoallelic expression patterns in bovine embryos and support recent results reported for ovine embryos. Collectively, the data suggest that monoallelic expression may not be required for most imprinted genes during preimplantation development, especially in ruminants. The research also suggests that monoallelic expression of genes may develop in a gene- and time-dependent manner.     

Establishing reference genes for use in real-time quantitative PCR analysis of early equine embryos

Real-time quantitative PCR (qPCR) is invaluable for investigating changes in gene expression during early development, since it can be performed on the limited quantities of mRNA contained in individual embryos. However, the reliability of this method depends on the use of validated stably expressed reference genes for accurate data normalisation. The aim of the present study was to identify and validate a set of reference genes suitable for studying gene expression during equine embryo development. The stable expression of four carefully selected reference genes and one developmentally regulated gene was examined by qPCR in equine in vivo embryos from morula to expanded blastocyst stage. SRP14, RPL4 and PGK1 were identified by geNorm analysis as stably expressed reference genes suitable for data normalisation. RPL13A expression was less stable and changed significantly during the period of development examined, rendering it unsuitable as a reference gene. As anticipated, CDX2 expression increased significantly during embryo development, supporting its possible role in trophectoderm specification in the horse. In summary, it was demonstrated that evidence-based selection of potential reference genes can reduce the number needed to validate stable expression in an experimental system; this is particularly useful when dealing with tissues that yield small amounts of mRNA. SRP14, RPL4 and PGK1 are stable reference genes suitable for normalising expression for genes of interest during in vivo morula to expanded blastocyst development of horse embryos.     

Angiopoietin-1 and -2 mRNA and protein expression in mouse preimplantation embryos and uteri suggests a role in angiogenesis during implantation

After attachment and migration through the endometrial epithelium, the embryo must induce angiogenesis within the endometrial stroma to successfully complete the implantation process. Growth factors have been shown to play an important role in embryo implantation and placentation. The aim of the study was to investigate the expression of angiopoietin-1 and -2 (Ang-1 and -2) mRNA and protein expression during the development of single preimplantation mouse embryos and of possible complementary expression in mouse uteri. Angiopoietin-1 mRNA was expressed throughout development in 78% of zygotes, 66% of 2-cell-embryos, 71% of 4-cell-embryos, 70% of 8-cell-embryos, 60% of morula stages, 48% of early blastocysts and 78% of late blastocysts. The number of Ang-1-expressing embryos in the early-blastocyst group was significantly different in comparison with zygotes, 4-cell-embryos, 8-cell-embryos and late blastocysts. Angiopoietin-2 mRNA and protein expression could not be detected in preimplantation embryos. Examination of the uteri revealed Ang-2 mRNA and protein expression in the oestrogen-dominated cycling phase and the progesterone-dominated mated phase, whereas Ang-1 expression was restricted to the mated phase. Herein, Ang-1 expression in preimplantation mouse embryos as well as Ang-1 and -2 expression in mouse uteri is demonstrated, suggesting a possible role for angiopoietins in the embryo-maternal dialogue of the implantation process via an enhancement of the vascular remodelling in favour of an implanting conceptus.     

RU 486 interferes with egg transport and retards the in vivo and in vitro development of mouse embryos

Oral administration of RU 486 at 100 mg/kg BW/day to mice on Days 1 and 2 of pregnancy caused retention of embryos in the oviduct and expulsion of those having entered the uterus. The treatment also retarded the development of embryos. In vitro study showed that RU 486 reduced the percentage of 2-cell mouse embryos developing into blastocysts. Thus, in addition to interfering with egg transport and impairing embryonic development , RU 486 can act directly on the embryo, interfering with preimplantation development .     

Potential and limitations of bovine-specific arrays for the analysis of mRNA levels in early development: preliminary analysis using a bovine embryonic array

New insights into the early development of large mammals are becoming available through the measurement of differential mRNA levels in oocytes and preimplantation embryos. These advances in knowledge are rapidly picking up in pace, mainly owing to the advantages brought by new molecular biology approaches being developed. The possibility of amplifying the starting material and therefore making measurements in single embryo units is now feasible. With these tools, the evaluation of variations in gene expression patterns during the preimplantation period or the impact of culture on mRNA levels is now possible. However, it is important to keep in mind that these methods still have limitations associated with sample preparation or the use of the appropriate controls. Even proper methods of analysis are very important to achieve the full benefit of the application of these tools. The present paper describes some of the potential, as well as limitations, of mRNA level analysis in early embryos, especially for microarray analysis. We have generated a bovine cDNA array (>2000 clones) that contains expressed sequence tags (ESTs) collected from various preimplantation development stages. Using this chip, we have initiated the characterisation of global mRNA level patterns of several key developmental stages from the immature oocyte to the blastocyst stage. As expected, the hybridisation results indicate very different expression profiles involving hundreds of genes when comparing oocyte and blastocyst samples to a reference mRNA sample made from a pool of ESTs from pooled somatic tissues. Although this array is still in its preliminary stage and the EST bank has not been processed to contain only unigenes, it is already a very useful tool for discovering candidate genes that may play important roles during early embryonic life.     

Does tributyl phosphate influence the radiation risk of a highly proliferating system--the early mouse embryo in vitro?

Two-cell mouse embryos were exposed in vitro to tributyl phosphate, x rays, or a combination of both. In-vitro development of the embryos was followed microscopically (cleavage to four- and eight-cell embryos, formation of morulae and blastocysts, and hatching of blastocysts). Effects on proliferation were estimated by counting the number of cells per embryo early (48 h p.c. = 48 hours post conceptionem) and late (144 h p.c.) in the preimplantation period. Cytogenetic damage was studied using micronucleus formation as the end point. Tributyl phosphate did not reveal toxic effects up to a concentration of about 5 microM after an exposure time of 18 h. At a concentration of about 15 microM, 50% of late preimplantation embryos showed effects on morphological development and on cell proliferation, and at about 40 microM, 90% of the embryos were affected. Tributyl phosphate did not induce micronuclei. Small effects by x irradiation were observed between 0.25 Gy and 0.5 Gy, depending on the end point measured in the late preimplantation stage. Fifty percent of the embryos were affected by a dose slightly higher than 1 Gy, and 90% after about 4 Gy. No enhancement in risk was found after combined treatment of the embryos with tributyl phosphate and x rays.     

In vivo gene transfer in mouse preimplantation embryos after intraoviductal injection of plasmid DNA and subsequent in vivo electroporation

The aim of this study was to investigate whether direct injection of nonviral DNA into the oviductal lumen and subsequent in vivo electroporation leads to in vivo gene transfer in mouse preimplantation embryos present within an oviduct, as an alternative to the pre-existing pronuclear microinjection-based transgenesis. With this technique, effects of expression of the gene of interest (GOI) on mouse preimplantation development can be monitored with relative ease. Superovulated 4-week-old B6C3F1 female mice (hybrids between C57BL/6N and C3H/HeN) were mated with adult B6C3F1 male mice. Two days later, females that had been identified as pregnant, based on the presence of copulation plugs, were injected with 1?μl of a solution containing an enhanced green fluorescent protein (EGFP) expression plasmid (0.5?μg) and 0.05% trypan blue. The entire oviduct was then electroporated using tweezer-type electrodes with 8 square-wave pulses of 50 V each with 50-ms duration. The next day, the 8-cell stage embryos were collected, and their number, morphology, and EGFP-derived fluorescence were recorded. Of the 12 oviducts (6 females used) examined, 3 contained fluorescent 8-cell stage embryos (33%, 19/58 tested), but the intensity of fluorescence varied among the embryos. In total, 10% (19/192 tested) of the embryos were fluorescent and the fluorescence was maintained in these embryos after 1 day of culture. However, the fluorescence disappeared in the late gestational stage fetuses, and the transgenes could not be detected. Our results indicate that it is possible to transfect in vivo preimplantation embryos, although the success rate appears to be relatively low and gene expression is transient. This technology may provide a new method for manipulating preimplantation embryos in vivo, by using, for example, Cre-mediated conditional DNA recombination.     

昆明鼠自体卵丘细胞与早期胚胎序贯共培养对胚胎体外发育潜能的影响

目的:评价昆明鼠胚胎与自体卵丘细胞序贯共培养对提高早期胚胎体外发育潜能的影响。方法:分两组进行实验,序贯共培养组使用分离培养的卵丘细胞与序贯培养液构建培养体系,与昆明鼠早期胚胎进行共培养;序贯培养组使用序贯培养液对早期胚胎进行序贯培养,观察两组胚胎各阶段囊胚形成率,检测囊胚总细胞数、囊胚细胞凋亡数,囊胚进一步培养获得的干细胞系数。结果:序贯共培养3d后总的囊胚形成率为68.13%,序贯培养组囊胚形成率为45.31%(P<0.05);囊胚总细胞数分别为76.52和38.57(P<0.05),细胞凋亡率分别为7.44%和16.28%(P<0.05)。将囊胚进一步培养,共培养组干细胞建系率为12.50%,序贯培养组未能建系。结论:昆明鼠胚胎与自体卵丘细胞序贯共培养与序贯培养比较,前者能更好地模拟体内环境,提高早期胚胎体外发育潜能。     

Epigenetic Aspects of Fertilization and Preimplantation Development in Mammals: Lessons from the Mouse

During gametogenesis, the parental genomes are separated and are epigenetically marked by modifications that will direct the expression profile of genes necessary for meiosis as well as for the formation of the oocyte and sperm cell. Immediately after sperm-egg fusion, the parental haploid genomes show great epigenetic asymmetry with differences in the levels of DNA methylation and histone tail modifications. The epigenetic program acquired during oogenesis and spermatogenesis must be reset for the zygote to successfully proceed through preimplantation development and this occurs as the two genomes approach each other in preparation for karyogamy. During preimplantation development, the embryo is vested with the responsibility of maintaining the primary imprints. In addition, female embryos must silence one of the X-chromosomes in order to transcribe equal levels of X-linked genes as their male counterparts. This review is intended as a survey of the epigenetic modifications and mechanisms present in zygotes and preimplantation mouse embryos, namely DNA methylation, histone modifications, dosage compensation, genomic imprinting, and regulation by non-coding RNAs.     

Preimplantation development in the streptozotocin-induced diabetic mouse

Streptozotocin (STZ) was used to develop a diabetic mouse model in which to study the development of the preimplantation embryo. STZ doses of 0, 160, 190, 210 and 240 mg kg-1 were given; 190 mg kg-1 was found to be the most suitable as the standard diabetogenic dose, providing about 60% mice with plasma glucose greater than 20 mM. The STZ-diabetic mice responded to superovulation with 10 i.u. of gonadotrophin in the same manner as control mice, producing similar embryo numbers at 48 h, 72 h and 96 h post-hCG. Furthermore, the proportion of 2-cell embryos collected from STZ-diabetic mice which developed to blastocysts in vitro was similar to that of 2-cell embryos from control mice. The STZ-diabetic mouse model after superovulation thus produced normal early preimplantation embryos whose development can be examined in detail in a diabetic environment.