Retrotransposon expression as a defining event of genome reprogramming in fertilized and cloned bovine embryos.

Genome reprogramming is the ability of a nucleus to modify its epigenetic characteristics and gene expression pattern when placed in a new environment. Low efficiency of mammalian cloning is attributed to the incomplete and aberrant nature of genome reprogramming after somatic cell nuclear transfer (SCNT) in oocytes. To date, the aspects of genome reprogramming critical for full-term development after SCNT remain poorly understood. To identify the key elements of this process, changes in gene expression during maternal-to-embryonic transition in normal bovine embryos and changes in gene expression between donor cells and SCNT embryos were compared using a new cDNA array dedicated to embryonic genome transcriptional activation in the bovine. Three groups of transcripts were mostly affected during somatic reprogramming: endogenous terminal repeat (LTR) retrotransposons and mitochondrial transcripts were up-regulated, while genes encoding ribosomal proteins were downregulated. These unexpected data demonstrate specific categories of transcripts most sensitive to somatic reprogramming and likely affecting viability of SCNT embryos. Importantly, massive transcriptional activation of LTR retrotransposons resulted in similar levels of their transcripts in SCNT and fertilized embryos. Taken together, these results open a new avenue in the quest to understand nuclear reprogramming driven by oocyte cytoplasm.

Reprogramming and differentiation in mammals: motifs and mechanisms.

The natural reprogramming of the mammalian egg and sperm genomes is an efficient process that takes place in less than 24 hours and gives rise to a totipotent zygote. Transfer of somatic nuclei to mammalian oocytes also leads to their reprogramming and formation of totipotent embryos, albeit very inefficiently and requiring an activation step. Reprogramming of differentiated cells to induced pluripotent stem (iPS) cells takes place during a period of time substantially longer than reprogramming of the egg and sperm nuclei and is significantly less efficient. The stochastic expression of endogenous proteins during this process would imply that controlled expression of specific proteins is crucial for reprogramming to take place. The fact that OCT4, NANOG, and SOX2 form the core components of the pluripotency circuitry would imply that control at the transcriptional level is important for reprogramming to iPS cells. In contradistinction, the much more efficient reprogramming of the mammalian egg and sperm genomes implies that other levels of control are necessary, such as chromatin remodeling, translational regulation, and efficient degradation of no longer needed proteins and RNAs.

Systems biology of the 2-cell mouse embryo.

The transcriptome of the 2-cell mouse embryo was analyzed to provide insight into the molecular networks at play during nuclear reprogramming and embryonic genome activation. Analysis of ESTs from a 2-cell cDNA library identified nearly 4,000 genes, over half of which have not been previously studied. Transcripts of mobile elements, especially those of LTR retrotransposons, are abundantly represented in 2-cell embryos, suggesting their possible role in introducing genomic variation, and epigenetic restructuring of the embryonic genome. Analysis of Gene Ontology of the 2-cell-stage expressed genes outlines the major biological processes that guide the oocyte-to-embryo transition. These results provide a foundation for understanding molecular control at the onset of mammalian development.

[The regulatory mechanisms of early embryogenesis in the mouse]. / Mekhanizmy reguliatsii rannego émbriogeneza myshi.

The mechanisms involved in the regulation of preimplantation mammalian development have been considered using the example of mouse embryos. The role of four factors affecting the program of early embryogenesis is discussed: nucleocytoplasmic interactions, "maternal" control of development, cell-to-cell interactions, and genomic imprinting. The current concepts of the spatial and temporal regulation of developmental processes have been reviewed, as well as the perspectives of some trends in the experimental embryology of mammals.

[The levels and nature of translation in mouse preimplantation embryos with repressed cytokinesis]. / Urovni i kharakter transliatsii u doimplantatsionnykh zarodyshei myshi s podavlennym tsitokinezom.

The translational activity of embryos cultured up to the 8-cell stage in cytochalasin D-supplemented medium was studied. These embryos remained unicellular during the entire preimplantation period. Blastocoel formation and hatching began in the cytochalasin-treated embryos at the same time as in the control. We have studied the overall translation rates of "one-cell embryos" and estimated the relative translational differences for individual polypeptides compared to the control embryos. Up to the early blastocyst stage, the translation levels in these embryos, measured by 35S-methionine incorporation, were about two times lower than in the control. At later stages, the differences were fivefold. The ppm values of approximately one-third of all individual spots on two-dimensional electrophoregrams differed three times or more between the control and cytochalasin-treated embryos. These results suggest that the first morphogenetic events in mammalian ontogenesis may be controlled autonomously and the timing of morphogenetic transitions is controlled by acquisition of a definite set of stage-specific factors which serves as the signal for subsequent development.

[Changes in the cavitation start time in chimeric CB6F1 <--> BALB/c embryos]. / Izmenenie vremeni zapuska kavitatsii u khimernykh zarodyshei CB6F1 <--> BALB/c.

It is found out that the acceleration of preimplantation morphogenesis is observed in mouse chimeras between single two-cell-stage blastomeres of BALB/c and [BALB/c x C57B1/6] x C57B1/6 (F2) embryos. The paired aggregation of blastomeres of two-cell stage made F2-BALB/c chimeras to start cavitation 8 +/- 2 hours earlier than F2<-->F2 and BALB/c-BALB/c control embryos. We have demonstrated for the first time the 'chimeric' heterosis on preimplantation stages. Possible approaches to the future studies of this phenomenon are discussed.

[The first and second polar bodies in mouse oogenesis]. / Izuchenie pervogo i vtorogo poliarnykh telets v oogeneze myshi.

Using light and electron microscopy, we studied several characteristics of the interphase nuclei and mitotic chromosomes in embryonic cells Cyclops kolensis (Lill) before and after chromatin diminution. We detected the reduction of one of the two nucleoli in somatic cells resulting from chromatin diminution. However, the ratio of the total diameter of the nucleoli to the diameter of the nucleus or to the diameter of the cell remains unchanged. Granules of the chromatin to be eliminated become visible in somatic cells of the C. kolensis embryos at the interphase of the fourth cleavage division. The structure of interphase nuclei and mitotic chromosomes of somatic cells of C. kolensis embryos is different before and after chromatin diminution. Possible mechanisms underlying these differences are discussed.

Preimplantation development of manipulated mouse zygotes fused with the second polar bodies: a cytogenetic study.

Immediately after fertilization one chromatid of each maternally-derived chromosome is extruded into the second polar body (2PB). We tested the ability of these "extra" chromosomes to support preimplantation development. Micromanipulation and electrofusion techniques were used to fuse 2PBs with diploid, haploid, or enucleated mouse zygotes. Androgenetic haploids, intact embryos, and digynic triploids served as the controls for the reconstructed embryos. Androgenetic haploid zygotes developed to the blastocyst stage only when fused with the 2PBs. This result demonstrates that even when extruded into the 2PB, chromosomes retain their ability to support normal preimplantation development. However, 2PB fusion with diploid zygote impaired preimplantation development. Normal development of experimentally produced digynic triploids (zygotes with one extra maternal pronucleus) indicated that developmental arrests, caused by the 2PB fusion, were not the result of triploidy or micromanipulation procedures. Cytogenetic studies showed that developmental failures of the reconstructed embryos were caused by premature chromosome condensation of the polar body chromosomes. This result indicates that 2PB must be removed from the zygotes' perivitelline space during animal cloning experiments. In addition, we showed that 2PB fusion with enucleated zygote is a reliable method for 2PB karyotyping and may be used in the studies of mammalian meiosis.