Vitrification of murine mature metaphase II oocytes perturbs DNA methylation reprogramming during preimplantation embryo development

Accurate reprogramming of DNA methylation occurring in preimplantation embryos is critical for normal development of both fetus and placenta. Environmental stresses imposed on oocytes usually cause the abnormal DNA methylation reprogramming of early embryos. However, whether oocyte vitrification alters the reprogramming of DNA methylation (5 mC) and its derivatives in mouse preimplantation embryo development remains largely unknown. Here, we found that the rate of cleavage and blastocyst formation of embryos produced by IVF of vitrified matured oocytes was significantly lower than that in control counterparts, but the quality of blastocysts was not impaired by oocyte vitrification. Additionally, although vitrification neither altered the dynamic changes of 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5 fC) before 4-cell stage nor affected the levels of 5 mC and 5-carboxylcytosine (5caC) throughout the preimplantation development, vitrification significantly reduced the levels of 5hmC and 5 fC from 8-cell stage onwards. Correspondingly, vitrification did not alter the expression patterns of Tet3 in preimplantation embryos but apparently reduced the expression levels of Tet1 in 4-cell and 8-cell embryos and increased the expression levels of Tet2 at morula stage. Taken together, these results demonstrate that oocyte vitrification perturbs DNA methylation reprogramming in mouse preimplantation embryo development.     

Measurement of metabolic turnover in single mouse embryos

The method for estimation of metabolic turnover by a single embryo at each stage of preimplantation development is based on the incubation of an embryo in the presence of labelled substrate at high specific activity in a miniaturized incubation chamber and the subsequent radioassay of metabolic products. Preliminary tests indicated that the treatment did not affect embryos adversely. Estimates of glycolysis, substrate oxidation and incorporation by mouse embryos throughout the whole of the preimplantation period of development were made. This technique could prove particularly useful for investigating substrate requirements and metabolic turnover in species for which few embryos are available for study.     

Dynamics of zonula occludens-2 expression during preimplantation embryonic development in the hamster

The objective was to study the expression of zonula occludens-2, a tight junction protein, during preimplantation hamster embryonic development, to predict its possible localization, source, and roles in trophectoderm differentiation and blastocyst formation in this species. Comparison of zonula occludens-2 expression pattern between the hamster and mouse preimplantation embryos from the zygote up to the blastocyst stage was also an objective of this study. Zonula occludens-2 localization was noted in nuclei of blastomeres in all stages of hamster and mouse embryonic development. Compared to mice, where zonula occludens-2 was first localized in the interblastomere membrane at the morula stage, hamster embryos had membranous zonula occludens-2 localization from the 2-cell stage onwards. Based on combined results of immunolocalization study in parthenogenic embryos and ovarian and epididymal sections, and quantitative PCR done in oocytes and all developmental stages of preimplantation embryos, perhaps there was a carry-over of zonula occludens-2 proteins or mRNA from the dam to the embryo. Based on these findings, we inferred that maternally derived zonula occludens-2 was involved in nuclear functions, as well as differentiation of blastomeres and blastocoel formation during preimplantation embryonic development in the hamster.     

Protein kinase Akt2/PKBβ is involved in blastomere proliferation of preimplantation mouse embryos

Activation of Akt/Protein Kinase B (PKB) by phosphatidylinositol-3-kinase (PI3K) controls several cellular functions largely studied in mammalian cells, including preimplantation embryos. We previously showed that early mouse embryos inherit active Akt from oocytes and that the intracellular localization of this enzyme at the two-cell stage depends on the T-cell leukemia/lymphoma 1 oncogenic protein, Tcl1. We have now investigated whether Akt isoforms, namely Akt1, Akt2 and Akt3, exert a specific role in blastomere proliferation during preimplantation embryo development. We show that, in contrast to other Akt family members, Akt2 enters male and female pronuclei of mouse preimplantation embryos at the late one-cell stage and thereafter maintains a nuclear localization during later embryo cleavage stages. Depleting one-cell embryos of single Akt family members by microinjecting Akt isoform-specific antibodies into wild-type zygotes, we observed that: (a) Akt2 is necessary for normal embryo progression through cleavage stages; and (b) the specific nuclear targeting of Akt2 in two-cell embryos depends on Tcl1. Our results indicate that preimplantation mouse embryos have a peculiar regulation of blastomere proliferation based on the activity of the Akt/PKB family member Akt2, which is mediated by the oncogenic protein Tcl1. Both Akt2 and Tcl1 are essential for early blastomere proliferation and embryo development.     

The development of preimplantation mouse parthenogenones in vitro in absence of glucose: Influence of the maternally inherited components

Mouse preimplantation embryo development is characterized by a switch from a dependence on the tricarboxylic acid cycle pre-compaction to a metabolism based on glycolysis post-compaction. In view of this, the role of glucose in embryo culture medium has come under increased analysis and has lead to improved development of outbred mouse embryos in glucose free medium. Another type of embryo that has proven difficult to culture is the parthenogenetic (PN) mouse embryo. With this in mind we have investigated the effect of glucose deprivation on PN embryo development in vitro. Haploid and diploid PN embryos were grown in medium M16 with or without glucose (M16-G) and development, glycolytic rate, and methionine incorporation rates assessed. Haploid PN and normal embryo development to the blastocyst stage did not differ in either M16 or M16-G. In contrast, although diploid PN embryos formed blastocysts in M16 (28.3%), they had difficulty in undergoing the morula/blastocyst transition in M16-G (7.6%). There was no significant difference in mean cell numbers of haploid PN, diploid PN and normal embryos cultured in M16 and M16-G at the morula and blastocyst stage. Transfer of diploid PN embryos from M16-G to M16 at the four- to eight-cell stage dramatically increased blastocyst development. At the morula stage diploid PN embryos grown in M16-G exhibited a higher glucose metabolism and protein synthesis compared to those grown in M16 and to haploid PN embryos. Difficulties of diploid PN embryos in undergoing the morula/blastocyst transition in absence of glucose infer the existence of a link between the maternally inherited components and the preimplantation embryos dependence on glucose. © 1996 Wiley-Liss, Inc.     

Buffalo rat liver cells produce factors that support preimplantation development of mouse embryos cultured in vitro

To examine the effects of buffalo rat liver (BRL) cells on the preimplantation development of mouse embryos in vitro, we first cultured two-cell mouse embryos alone in serum-free Dulbecco modified Eagle medium. As expected, the embryos did not develop to subsequent stages. However, when cocultured with BRL cells, the embryos developed to the blastocyst stage efficiently. Direct contact of embryos with BRL cells was not necessary for development: the medium conditioned by BRL cells contained soluble factors that supported the preimplantation development of mouse embryos. Embryos cultured with BRL-conditioned medium that was replaced at various intervals had a further increased rate of development to the blastocyst stage. This finding indicated that the activities of the factors were maintained only briefly. Seven proteins between 35 and 44 kDa that were detected in the medium were highly beneficial to the development of the embryos. Follistatin-related protein and pigment epithelium-derived factor are believed to be the factors supporting embryo development. The other five proteins also may improve the environment for the development of mouse embryos cultured in vitro.     

Serotonin localization and its functional significance during mouse preimplantation embryo development

Serotonin is a neurotransmitter functioning also as a hormone and growth factor. To further investigate the biological role of serotonin during embryo development, we analysed serotonin localization as well as the expression of specific serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos. The functional significance of serotonin during the preimplantation period was examined by studying the effects of serotonin on mouse embryo development. Embryo exposure to serotonin (1 microM) highly significantly reduced the mean cell number, whereas lower concentrations of serotonin (0.1 microM and 0.01 microM) had no significant effects on embryo cell numbers. In all serotonin-treated groups a significant increase in the number of embryos with apoptotic and secondary necrotic nuclei was observed. Expression of serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos was confirmed by in situ hybridization showing a clearly distinct punctate signal. Immunocytochemistry results revealed the localization of serotonin in oocytes and embryos to the blastocyst stage as diffuse punctate cytoplasmic labelling. It appears that endogenous and/or exogenous serotonin in preimplantation embryos could be involved in complex autocrine/paracrine regulations of embryo development and embryo-maternal interactions.     

Abnormal development and transport and increased sister-chromatid exchange in preimplantation embryos following superovulation in mice

Both sister-chromatid exchange (SCE) response and embryonic development and transport in preimplantation embryos were evaluated on day 3 of gestation (vaginal plug = 1) of superovulated Swiss mice. Superovulation was found to have significant effects on number of preimplantation embryos (increase), embryo localization (accelerated transport), cleavage rate (advanced development) and abnormality rate (misshaped, fragmented, dead embryos). Superovulated 4- and 8-cell embryos collected from oviducts and uteri and incubated in vitro with 5-bromodeoxyuridine (BrdU) displayed up to 4 times higher SCE frequency than spontaneously ovulated embryos. This increase is independent of stage of development and location at the time of embryo collection. The results indicate that superovulated embryos may have induced DNA lesions.     

Expression of beta-galactosidase in preimplantation ovine and porcine embryos

Knowledge regarding the timing of embryonic expression of the mammalian genome is of relevance for the development of preimplantation diagnostic methods for human genetic diseases. For development of preimplantation diagnosis of lysosomal storage diseases, it will be necessary to know at which embryonic stage the genes for lysosomal enzymes are expressed. In previous studies by other investigators, it has been shown that lysosomal alpha- and beta-galactosidase and beta-glucuronidase in murine embryos increase 50- to 100-fold in activity between the two-cell and late blastocyst stage. We describe here expression of lysosomal beta-galactosidase in preimplantation ovine (two-cell through midblastocyst) and porcine (two-cell through late blastocyst) embryos. Expression of beta-galactosidase in ovine and porcine preimplantation embryos followed a similar rate of increase as that described for murine embryos. Activity of beta-galactosidase increased over 10-fold between the two- to four-cell and midblastocyst stages in ovine embryos, and 300-fold between the two- to four-cell and late blastocyst stages in porcine embryos. Activity expressed on a per cell basis was relatively constant in ovine embryos, as has been described in murine embryos, and increased approximately 5-fold on a per cell basis in porcine embryos. Activity of beta-galactosidase in ovine and porcine embryos initially was greater than 12-fold on a per cell or per embryo basis than in murine embryos evaluated. The knowledge of beta-galactosidase embryonic expression may provide the basis for preimplantation diagnosis of genetic beta-galactosidase deficiency in these species.     

Role of glucose in mouse preimplantation embryo development

Mouse preimplantation embryos consume pyruvate preferentially during the early developmental stages, before glucose becomes the predominant energy substrate in the blastocyst. To investigate the importance of the switch to glucose utilization at the later developmental stages, mouse embryos from F1 hybrid mice (CBA/Ca × C57BL/6) were cultured from the one-and two-cell stages (22 and 46 h post hCG, respectively) for 5 days in a modified medium, M16, containing 0.33 mM pyruvate and 5 or 23 mM D+L-lactate, in the presence and absence of 1 mM glucose (M16+G and M16-G, respectively). Nutrient uptakes were also determined over this time. Some embryos cultured in M16-G were transferred to M16+G at 94 or 118 h post hCG. Embryos cultured from the two-cell stage in M16+G exhibited the characteristic fall in pyruvate consumption between the morula and the blastocyst stage; those cultured from the two-cell stage in M16-G compensated for the lack of glucose by consuming increasing amounts of pyruvate, from 2.78 pmol/embryo/h at 58 h post hCG to 5.21 pmol/embryo/h at 154 h post hCG. However, the percentage of embryos developing to the blastocyst stage, the hatching rate, and blastocyst cell numbers (50.6 ± 2.5 [28] vs. 105 ± 3.8 [37]) were all lower in this group. When exposed to glucose at 94 or 118 h post hCG, embryos cultured from the two-cell stage in M16-G readily consumed glucose in preference to pyruvate, although the characteristic fall in pyruvate consumption was not observed. One-cell embryos cultured continuously in M16-G were only able to develop to the morula stage, after which time they degenerated. In these embryos pyruvate was readily consumed between 22 and 94 h post hCG, before falling from 2.77 pmol/embryo/h at 83 h post hCG to 0.045 pmol/embryo/h at 130 h post hCG. Transfer of these embryos to M16+G at 94 and 118 h post hCG did not support development to the hatching blastocyst stage. The results show that mouse preimplantation embryos from F1 hybrid mice (CBA/Ca × C57BL/6) need only be exposed to glucose for less than 24 h between 22 and 94 h post hCG in order to develop from the morula to the blastocyst stage in vitro. However, the exposure time needs to be increased to between 24 and 72 h in order that blastocyst cell numbers reach control levels. The importance of glucose before the morula stage may relate to the need to synthesize glycogen for later use. If the obligatory requirement for glucose is fulfilled, embryos are able to utilize pyruvate in the absence of glucose at the later stages of development. These results show that the mouse preimplantation embryo can, to some extent, adapt metabolically to changes in its external environment. © 1995 Wiley-Liss, Inc.     

Precise recapitulation of methylation change in early cloned embryos

Change of DNA methylation during preimplantation development is very dynamic, which brings this term to the most attractive experimental target for measuring the capability of cloned embryo to reprogram its somatic genome. However, one weak point is that the preimplantation stage carries little information on genomic sequences showing a site-specific re-methylation after global demethylation; these sequences, if any, may serve as an advanced subject to test how exactly the reprogramming/programming process is recapitulated in early cloned embryos. Here, we report a unique DNA methylation change occurring at bovine neuropeptide galanin gene sequence. The galanin gene sequence in early bovine embryos derived by in vitro fertilization (IVF) maintained a undermethylated status till the morula stage. By the blastocyst, certain CpG sites became methylated specifically, which may be an epigenetic sign for the galanin gene to start a differentiation programme. The same sequence was moderately methylated in somatic donor cell and, after transplanted into an enucleated oocyte by nuclear transfer (NT), came rapidly demethylated to a completion, and then, at the blastocyst stage, re-methylated at exactly the same CpG sites, as observed so in normal blastocysts. The precise recapitulation of normal methylation reprogramming and programming at the galanin gene sequence in bovine cloned embryos gives a cue for the potential of cloned embryo to superintend the epigenetic states of foreign genome, even after global demethylation.     

Effect of polyamine limitation on DNA synthesis and development of mouse preimplantation embryos in vitro

In-vitro treatment of preimplantation mouse embryos with spermine and spermidine biosynthesis inhibitor, methylglyoxal-bis-(guanylhydrazone) (MGBG), arrested embryo development at the 8-cell or morula stage. In addition, the embryo DNA synthetic rate, as measured by [3H]thymidine incorporation, was strongly inhibited. The inhibition of blastocyst formation and DNA synthesis by MGBG was readily reversible by an exogenous supply of spermine and/or spermidine to the culture medium. DL-alpha-Methylornithine or DL-alpha-difluoromethylornithine (alpha-DFMO), inhibitors of putrescine biosynthesis, had no effect on embryos cultured for 1 or 2 days, but on the 3rd day embryo DNA synthesis was significantly depressed in the presence of alpha-DFMO. These observations suggest that, during early development of the preimplantation mouse embryo, spermine and spermidine are involved in regulation of embryo growth and DNA synthesis. They may also indicate a role of putrescine at a later stage of mouse embryo development.     

Levels and modification of methionyl-transfer RNA in preimplantation rabbit embryos.

Transfer RNA with l-methionine acceptor activity was extracted from preimplantation rabbit embryos and purified on reverse-phase-3 columns. The molar quantity of methionine acylated to RNA increases as embryo development proceeds from the 16-cell stage to the 80,000 cell blastocyst stage. However, the quantity of methionyl-tRNA per genome declines 100-fold as the embryo cell number increases. Formylation of methionyl-tRNA illustrated that approximately one-third of tRNA^Met extracted was tRNA_f^Met. Methylation of purified methionyl-tRNA by an adult rabbit liver methylase extract illustrated that two-day preimplantation embryo tRNA is highly hypomethylated relative to tRNA from later stages of development. The hypomethylated methionyl-tRNA was also less effective in ribosome binding studies than more fully methylated methionyl-tRNA present in the later stages of embryo development.