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MII-arrested rat oocytes spontaneously resume meiotic maturation soon after recovery from oviducts. Time elapsed post oocyte retrieval, environmental factors, certain genetic background have been implied to promote spontaneous release from MII arrest in rat oocytes. The precise mechanism behind this process is unknown. This study was undertaken to explore signaling pathways, which may be involved in spontaneous activation in rat oocytes. Using triple immuno-staining and epifluorescence microscopy we have described morphological changes in the meiotic spindle of rat oocytes during 6h of in vitroculture (immature CD female rats from Charles River Laboratories). An ELISA-based method was used to evaluate cdc2 activity in rat oocytes subjected to in vitroculture. SDS-PAGE and western blotting were performed to study levels of phosphorylated and total p42/p44 MAPK, phosphorylated on Tyr15 and total cdc2, cyclin B1 and β actin. Expression of Emi2 was analysed by RT-PCR. Data were analyzed using GLMM or ANOVA followed by t-test. Freshly collected oocytes contained well-preserved spindle with chromosomes aligned in metaphase plate. After being in culture for 2 h oocytes demonstrated signs of activation, such as spindle rotation, preparing to extrude second polar body, and some oocytes had entered anaphase. In the majority of oocytes cultured for 6 h spindles had disintegrated and chromosomes were scattered in the oocyte cytoplasm; microtubules were found around condensed chromosomes. Significant drop in cdc2 activity was detected in oocytes after 2 h of in vitroculture. In oocytes cultured for additional 4h of cdc2 activity returned to the level observed in freshly collected oocytes. Inhibitory phosphorylation of cdc2 on Tyr 15 was not associated with cdc2 inactivation. We were not able to detect changes in the level of cyclin B1 during the MII to MIII transition. Phosphorylated forms of activated p42/p44 MAPK in eggs were present throughout in vitroculture. Emi2 is a novel candidate cytostatic factor in vertebrate eggs. Expression of Emi2 was detected at mRNA level in rat eggs and zygotes with no expression at later stages of preimplantation development. Taken together, the onset of spontaneous activation in rat oocytes is correlated with a sharp decline in cdc2 activity and stable level of phosphorylated p42/p44 MAPK. Dynamics of cdc2 and p42/p44 MAPK activity during spontaneous activation resembles that during the MI to MII transition, although spontaneously activated rat oocytes do not form MIII spindle. Molecular factors involved in spindle assembly may be missing during the MII to MIII transition. Persistence of p42/p44 MAPK in spontaneously activated rat oocytes could account for the absence of pronuclear formation. The role of Emi2 remains to be investigated.This work is supported by the CMVM, ORS, Mary Orr Paterson studentships and the Framework 6 activity EURATOOLS. [ABSTRACT FROM AUTHOR]

Mammalian eggs await fertilisation while arrested at the second metaphase stage of meiotic division. A network of signalling pathways enables the establishment and maintenance of this metaphase-II arrest. In the absence of fertilisation, mammalian eggs can spontaneously exit metaphase II when parthenogenetically stimulated, or sometimes without any obvious stimulation. Ovulated rat eggs abortively release from metaphase-II arrest once removed from egg donors. Spontaneously activated rat eggs extrude the second polar body and proceed to the so-called metaphase III-'like' stage, with clumps of condensed chromatin scattered in the egg cytoplasm. It is still unclear what makes rat eggs susceptible to spontaneous activation; however, a vague picture of the signalling pathways involved in the process of spontaneous activation is beginning to emerge. Such cell cycle instability is one of the major reasons why it is more difficult to establish nuclear transfer in the rat. This review examines the known predisposing factors and biochemical mechanisms involved in spontaneous activation. The strategies used to prevent spontaneous metaphase-II release in rat eggs will also be discussed., (Copyright © 2011 Wiley Periodicals, Inc.)

Methylation of cytosine is a DNA modification associated with gene repression. Recently, a novel cytosine modification, 5-hydroxymethylcytosine (5-hmC) has been discovered. Here we examine 5-hmC distribution during mammalian development and in cellular systems, and show that the developmental dynamics of 5-hmC are different from those of 5-methylcytosine (5-mC); in particular 5-hmC is enriched in embryonic contexts compared to adult tissues. A detectable 5-hmC signal appears in pre-implantation development starting at the zygote stage, where the paternal genome is subjected to a genome-wide hydroxylation of 5-mC, which precisely coincides with the loss of the 5-mC signal in the paternal pronucleus. Levels of 5-hmC are high in cells of the inner cell mass in blastocysts, and the modification colocalises with nestin-expressing cell populations in mouse post-implantation embryos. Compared to other adult mammalian organs, 5-hmC is strongly enriched in bone marrow and brain, wherein high 5-hmC content is a feature of both neuronal progenitors and post-mitotic neurons. We show that high levels of 5-hmC are not only present in mouse and human embryonic stem cells (ESCs) and lost during differentiation, as has been reported previously, but also reappear during the generation of induced pluripotent stem cells; thus 5-hmC enrichment correlates with a pluripotent cell state. Our findings suggest that apart from the cells of neuronal lineages, high levels of genomic 5-hmC are an epigenetic feature of embryonic cell populations and cellular pluri- and multi-lineage potency. To our knowledge, 5-hmC represents the first epigenetic modification of DNA discovered whose enrichment is so cell-type specific., (© 2011 IBCB, SIBS, CAS All rights reserved)

In the present study, some modifications were made to the zona-free nuclear transfer technique in the mouse in order to achieve greater efficiency. Firstly, a 1-h interval was allowed between cumulus removal and zona pellucida digestion. Secondly, acid Tyrode's was selected for zona pellucida removal, because contrary to pronase, it allows embryo survival during parthenogenic activation in the absence of calcium. Even when the exposure time to pronase was reduced to as little as 1 min or washed with fetal calf serum to inhibit the enzyme, the percentage of lysis during activation in the absence of calcium was still very high. Thirdly, electrofusion was performed at room temperature (21 degrees C), instead of 30 degrees C as in our previous experiments. Finally, embryos were cultured in groups of 12-15, instead of individually, using a "well of the wells" system during activation and culture. When compared, parthenogenic activated control embryos showed an increase in the development to blastocyst when cultured in pairs instead of individually. By the end of the experiments and using embryonic stem (ES) cells, there was a significant increase in fusion rate (1.5-fold increase) and in development to morula/blastocyst from cleaved reconstructed embryos (1.5-fold increase) when compared with the results before the modifications. A 2.4-fold increase in overall efficiency was achieved from the oocyte to morula/blastocyst stages.

In the present study, a zona-free nuclear transfer (NT) technique, which had been originally developed in cattle, was modified for the mouse. Steps involved in this approach include removing the zona pellucida and enucleating without a holding pipette; sticking donor cells to the cytoplast before electric pulses are applied to fuse them and culturing reconstructed embryos individually in single droplets, to prevent aggregation. Control zona-free and zona-intact embryos from mated donors showed no significant difference in development to blastocyst, but did show reduced development to term. Removal of the zona pellucida affected the response to activation by strontium in the absence of calcium as a significant proportion of zona-free control oocytes and embryos reconstructed by NT lysed during this treatment. A comparison between cumulus and ES cells as donor cells revealed significant differences in fusion efficiency (58.1 +/- 4.0%, n = 573 vs. 42.9 +/- 2.2%, n = 2064, respectively, p < 0.001), cleavage (77.2 +/- 3.4%, n = 334 vs. 40.8 +/- 2.7%, n = 903, respectively, p < 0.001) but not for development to morula/blastocyst (8.7 +/- 2.1%, n = 334 vs. 13.9 +/- 1.8%, n = 903, respectively, p < 0.1). The stage at which embryo development arrested was also affected by donor cell type. A majority of embryos reconstructed from cumulus cells arrested at two-cell stage, usually with two nuclei, whereas those reconstructed from ES cells arrested at one-cell stage, usually with two pseudo-pronuclei. After transfer of ES cell-derived NT embryos, a viable cloned mouse was produced (3.0% of transferred embryos developed to term). These observations establish that a zona-free cloning approach is possible in the mouse, although further research is required to increase the efficiency.