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Nucleolar remodeling in nuclear transfer embryos.: Adv Exp Med Biol, Vol. 591 (2007), pp. 84-92.Transcri ption of the ribosomal RNA (rRNA) genes occurs in the nucleolus and results in ribosome biogenesis. The rRNA gene activation and the associated nucleolus formation may be used as a marker for the activation of the embryonic genome in mammalian embryos and, thus serve to evaluate the developmental potential of embryos originating from varied nuclear transfer protocols. In bovine in vivo developed embryos, functional ribosome-synth esizing nucleoli become structurally distinct toward the end of the 4th post-fertiliza tion cell cycle. In embryonic cell nuclear transfer embryos, fully developed nucleoli are not apparent until the 5th cell cycle, whereas in somatic cell nuclear transfer embryos the functional nucleoli emerge already during the 3rd cell cycle. Intergeneric reconstructed embryos produced by the fusion of bovine differentiated somatic cell to a nonactivated ovine cytoplast fail to develop fully functional nucleoli. In bovine in vivo developed embryos, a range of important nucleolar proteins (e.g., topoisomerase I, upstream binding factor and RNA polymerase I, fibrillarin, nucleophosmin and nucleolin) become localized to the nucleolar anlage over several cell cycles. This relocation is completed toward the end of the 4th cell cycle. A substantial proportion of bovine embryos produced by nuclear transfer of embryonic or somatic cells to bovine ooplasts display aberrations in protein localization in one or more blastomers. This information is indicative of underlying aberrations in genomic reprogramming and may help to explain the abnormalities observed in a proportion of fetuses and offspring derive from nuclear transfer embryos.J Laurincik, P Maddox-Hyttel

Source: Adv Exp Med Biol, Vol. 591 (2007), pp. 84-92.
Epigenetic programming in the preimplantatio n rat embryo is disrupted by chronic paternal cyclophosphami de exposure.: Proc Natl Acad Sci U S A, Vol. 102, No. 22. (31 May 2005), pp. 7865-7870.Prec onceptional paternal exposure to cyclophosphami de, a widely used anticancer agent, leads to increases in embryo loss, malformations, and behavioral deficits in offspring; these abnormalities are transmissible to subsequent generations [Auroux, M., Dulioust, E., Selva, J. & Rince, P. (1990) Mutat. Res. 229, 189-200]. Little information exists on the mechanisms underlying this male-mediated developmental toxicity. We assessed the impact of paternal cyclophosphami de exposure on the dynamic regulation of histone H4 acetylation at lysine 5 and DNA methylation in preimplantatio n rat embryos. Zygotes sired by drug-treated males displayed advanced developmental progression, increased pronuclear areas, and disruption of the epigenetic programming of both parental genomes. Early postfertilizat ion zygotic pronuclei were hyperacetylate d; by mid-zygotic development, male pronuclei were dramatically hypomethylated , whereas female pronuclei were hypermethylate d. Micronuclei were substantially elevated, and histone H4 acetylation at lysine 5 localization to the nuclear periphery was disrupted in two-cell embryos fertilized by cyclophosphami de-exposed spermatozoa. This finding demonstrates that paternal exposure to this drug induces aberrant epigenetic programming in early embryos. We hypothesize that disturbances in epigenetic programming contribute to heritable instabilities later in development, emphasizing the importance of epigenetic risk assessment after chemotherapy.T S Barton, B Robaire, BF Hales

Source: Proc Natl Acad Sci U S A, Vol. 102, No. 22. (31 May 2005), pp. 7865-7870.
Ethanol elevates c-Myc levels in cultured mouse preimplantatio n embryos.: Alcohol Clin Exp Res, Vol. 23, No. 5. (May 1999), pp. 778-784.A brief exposure to ethanol accelerates the rate of early mouse embryonic development in vitro, increasing blastocyst formation, trophoblast outgrowth, and implantation rates after embryo transfer. The physiological effects of ethanol during preimplantatio n development are associated with rapid changes in gene expression and apparently arise from the ability of ethanol to elevate cytoplasmic free Ca2+ and alter cellular signaling pathways. The purpose of this study was to examine whether the abundance of c-Myc, a transcription factor that promotes cell proliferation and is required for blastocyst development, is upregulated in mouse blastocysts challenged with ethanol. After exposure of mouse blastocysts to 0.1% (17.5 mM) ethanol, wc determined the levels of: 1) c-Myc mRNA, using reverse transcription and the polymerase chain reaction; and 2) c-Myc protein levels, using specific monoclonal antibodies. Within 10 min of exposure to ethanol, the relative abundance of c-Myc mRNA increased 6-fold, then rapidly returned to baseline levels within 1 hr. As expected, elevation of c-Myc mRNA by ethanol was attenuated in embryos that were first treated with the intracellular Ca2+ chelator, BAPTA-AM. Western blot analysis of solubilized embryos revealed that c-Myc mRNA was translated into a single 62-kD protein that increased in intensity 30 min after treatment with ethanol. Immunocytochem ical staining demonstrated that c-Myc was localized exclusively in nuclei and that staining intensity increased significantly after 10 min. Peak levels of c-Myc protein were found 30 min after ethanol exposure and persisted for at least 2 hr. The c-myc proto-oncogene seems to be an immediate early response gene for ethanol that may regulate the transcription of other genes that influence early embryogenesis and growth.RE Leach, UK Rout, JF Schultz, DE Saunders, DR Armant

Source: Alcohol Clin Exp Res, Vol. 23, No. 5. (May 1999), pp. 778-784.
An intact sperm nuclear matrix may be necessary for the mouse paternal genome to participate in embryonic development.: Biol Reprod, Vol. 60, No. 3. (March 1999), pp. 702-706.We have been interested in determining the minimally required elements in the sperm head that are necessary in order for the paternal genome to participate in embryogenesis. We used an ionic detergent, mixed alkyltrimethyl ammonium bromide (ATAB), plus dithiothreitol (DTT) to remove the acrosome and almost all of the perinuclear theca, leaving only the sperm nucleus morphologicall y intact. We also tested the stability of the sperm nuclear matrix by the ability to form nuclear halos. Sperm nuclei washed in freshly prepared 0.5% ATAB + 2 mM DTT completely decondensed when extracted with salt, but nuclei washed in the same buffer that was 1 wk old, and then extracted with salt, produced nuclear halos, indicating stable nuclear matrices. When we treated sperm heads with freshly prepared ATAB+DTT and injected them into oocytes, none of the oocytes developed into live offspring. In contrast, sperm heads treated in the same way but with 1-wk-old ATAB+DTT solution could support development of about 30% of the oocytes to live offspring. Electron microscopy demonstrated that most of the perinuclear theca had been removed in both cases. These data suggest that at least in the mouse, the only component of the spermatozoa that is crucial for participation in embryologic development is the sperm nucleus with a stable nuclear matrix.WS Ward, Y Kimura, R Yanagimachi

Source: Biol Reprod, Vol. 60, No. 3. (March 1999), pp. 702-706.
Paternal exposure to cyclophosphami de dysregulates the gene activation program in rat preimplantatio n embryos.: Mol Reprod Dev, Vol. 57, No. 3. (November 2000), pp. 214-223.Althou gh there has been progress in determining the mechanisms by which maternal toxicant exposure affects progeny, there is little information on the actions of drugs administered to the father. We investigated the effects of pre-conception al paternal exposure to cyclophosphami de, an anti-cancer agent, on embryonic gene activation in the rat. The male pronucleus was formed earlier in embryos sired by cyclophosphami de-treated male rats than in those sired by controls; early male pronucleus formation was followed by alterations in the gene activation program. BrUTP incorporation into RNA and Sp1 transcription factor immunostaining were increased and spread over both cytoplasmic and nuclear compartments in 2-cell embryos sired by cyclophosphami de-treated males compared to controls. Total RNA synthesis was constant in 1-8 cell embryos sired by drug-treated fathers, while in control embryos RNA synthesis increased four-fold to peak at the 4-cell stage. In 2-cell embryos sired by drug-treated males, the relative abundance of candidate imprinted genes was elevated significantly above control; a peak in the expression of these genes was not observed until the 8-cell stage in control embryos. Thus, paternal drug exposure temporally and spatially dysregulated rat zygotic gene activation, altering the developmental clock.W Harrouk, S Khatabaksh, B Robaire, BF Hales

Source: Mol Reprod Dev, Vol. 57, No. 3. (November 2000), pp. 214-223.
Embryonic genome activation.: Front Biosci, Vol. 6 (1 June 2001)Genome activation is one of the first critical events in the life of the new organism. Both the timing of genome activation and the array of genes activated must be controlled correctly. Genome activation occurs in a stepwise manner, with some genes being transcribed well in advance of the major genome activation event, in which most housekeeping genes become activated. Changes in chromatin protein content, particularly histone proteins, and chromatin structure appear to regulate the availability of the genome for transcription and provide for specificity of transcription. Gene enhancers are not initially required for transcription, but become necessary as the chromatin structure is modified. Changes in transcription factor content or activity are also required, and protein synthesis is essential for genome activation during both early and later phases of transcriptiona l activation. Both the changes in chromatin structure and availability of transcription factors are regulated by cell cycle-dependen t mechanisms, thus providing the necessary coordination between these processes and other processes such as DNA replication and cleavage.KE Latham, RM Schultz

Source: Front Biosci, Vol. 6 (1 June 2001)
Expression of proto-oncogene s in mouse eggs and preimplantatio n embryos.: Mol Reprod Dev, Vol. 35, No. 1. (May 1993), pp. 8-15.The expression of several protooncogenes has been investigated in mouse eggs and preimplantatio n embryos using reverse transcription coupled to amplification of cDNAs by the polymerase chain reaction (RT-PCR). The genes chosen for analysis included both cytoplasmic (c-raf-1, rasH, rasK, and rasN) and nuclear (c-fos and c-myc) proto-oncogene s encoding proteins involved in the transduction of signals from protein-tyrosi ne kinase growth factor receptors. Transcripts of the cytoplasmic proto-oncogene s were detected both as maternal and embryonic mRNAs at levels (ca. 1,000 copies per egg or embryo) approximately comparable to their levels of transcription in somatic cells. Transcripts of c-fos and c-myc were also detected in both eggs and embryos, although at more variable levels: Maternal transcripts were present at very low levels (ca. 1-10 copies per egg) in growing oocytes and ovulated eggs; embryonic transcription of c-myc increased, reaching mRNA levels of approximately 100-1,000 copies per embryo in four-cell embryos, morula, and blastocysts; in contrast the transcription of c-fos remained at low, barely detectable levels throughout preimplantatio n development. Although the significance of the low levels of c-fos mRNA is unclear, these results indicate that preimplantatio n embryos possess the basic intracellular signaling apparatus required to respond to polypeptide growth factors.SK Pal, R Crowell, AA Kiessling, GM Cooper

Source: Mol Reprod Dev, Vol. 35, No. 1. (May 1993), pp. 8-15.
The stress response in gametes and embryos after paternal chemical exposures: Toxicology and Applied Pharmacology, Vol. 207, No. 2, Supplement 1. (1 September 2005), pp. 514-520.There is increasing concern that paternal exposure to toxic chemicals impacts negatively on progeny outcome. Exposure of male rats to a model male-mediated developmental toxicant and anticancer alkylating agent, cyclophosphami de, resulted in increased pre- and post-implantat ion loss, as well as in malformations. We hypothesize that the stage specificity of the effects of paternal cyclophosphami de exposure on progeny depends on the ability of germ cells to respond to stress, repair DNA or undergo apoptosis. Acute high dose exposure of male rats to cyclophosphami de increased the expression of heat shock proteins and DNA repair genes, predominantly in round spermatids. In contrast, chronic low dose treatment dramatically decreased the expression of stress response genes in pachytene spermatocytes and round spermatids, but not in elongated spermatids; this reduced ability to respond to stress may allow damage to accumulate, resulting in altered sperm function. Increased DNA damage was maximal 3 weeks after drug exposure, during spermiogenesis , a key point in sperm chromatin remodelling. Drug exposure for 9 weeks increased the frequency of spermatozoa with chromosome 4 disomy and nullisomy. DNA damage found in cyclophosphami de-exposed spermatozoa was imparted to the newly fertilized zygote. Drug-exposed spermatozoa decondensed more rapidly than control spermatozoa and male pronuclear formation was earlier. RNA synthesis was higher in 1-cell embryos sired by drug-treated fathers than in controls. Significantly, the profile of gene expression was altered in embryos sired by drug-treated males as early as the 1-cell stage. Thus, exposure of male rats to cyclophosphami de altered male germ cell quality with a consequent temporal and spatial disruption of the zygotic genome activation.Bar bara Hales, Adriana Aguilar-Mahech a, Bernard Robaire

Source: Toxicology and Applied Pharmacology, Vol. 207, No. 2, Supplement 1. (1 September 2005), pp. 514-520.
Histone arginine methylation regulates pluripotency in the early mouse embryo: Nature, Vol. 445, No. 7124., pp. 214-218.Maria- Elena Torres-Padilla , David-Emlyn Parfitt, Tony Kouzarides, Magdalena Zernicka-Goetz

Source: Nature, Vol. 445, No. 7124., pp. 214-218.
Aberrant lamination in the cerebral cortex of mouse embryos lacking DNA topoisomerase IIbeta.: Proc Natl Acad Sci U S A, Vol. 100, No. 12. (10 June 2003), pp. 7123-7128.We have examined corticogenesis in mouse embryos lacking DNA topoisomerase IIbeta (IIbeta) in the brain or in all tissues. The absence of IIbeta, a type II DNA topoisomerase normally expressed in postmitotic cells in the developing cortex, severely affects cerebral stratification : no subplate is discernible, and neurons born at later stages of corticogenesis fail to migrate to the superficial layers. This abnormal pattern of neuron positioning in the cerebral cortex is reminiscent of that observed in mouse mutants defective in the reelin-signali ng pathway. Significantly, the level of reelin in the neocortex is much reduced when IIbeta is absent. These results implicate a role of IIbeta in brain development. The enzyme may be required in implementing particular genetic programs in postmitotic cells, such as reelin expression in Cajal-Retzius cells, perhaps through its action on nucleoprotein structure of particular chromosomal regions.YL Lyu, JC Wang

Source: Proc Natl Acad Sci U S A, Vol. 100, No. 12. (10 June 2003), pp. 7123-7128.

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