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Embryo manipulation via assisted reproductive technology and epigenetic asymmetry in mammalian early development.

Kohda T, Ishino F - Philos. Trans. R. Soc. Lond., B, Biol. Sci. (2013)

Bottom Line: The sperm and egg DNA methylation profiles are very different from each other, and just after fertilization, only the paternally derived genome is subjected to genome-wide hydroxylation of 5-methylcytosine, resulting in an epigenetic asymmetry in parentally derived genomes.Zygotic gene activation from paternally or maternally derived genomes also starts around the two-cell stage, presumably in a different manner in each of them.In particular, we focus on the effects of intracytoplasmic sperm injection that can result in long-lasting transcriptome disturbances, at least in mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Epigenetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.

ABSTRACT
The early stage of mammalian development from fertilization to implantation is a period when global and differential changes in the epigenetic landscape occur in paternally and maternally derived genomes, respectively. The sperm and egg DNA methylation profiles are very different from each other, and just after fertilization, only the paternally derived genome is subjected to genome-wide hydroxylation of 5-methylcytosine, resulting in an epigenetic asymmetry in parentally derived genomes. Although most of these differences are not present by the blastocyst stage, presumably due to passive demethylation, the maintenance of genomic imprinting memory and X chromosome inactivation in this stage are of critical importance for post-implantation development. Zygotic gene activation from paternally or maternally derived genomes also starts around the two-cell stage, presumably in a different manner in each of them. It is during this period that embryo manipulation, including assisted reproductive technology, is normally performed; so it is critically important to determine whether embryo manipulation procedures increase developmental risks by disturbing subsequent gene expression during the embryonic and/or neonatal development stages. In this review, we discuss the effects of various embryo manipulation procedures applied at the fertilization stage in relation to the epigenetic asymmetry in pre-implantation development. In particular, we focus on the effects of intracytoplasmic sperm injection that can result in long-lasting transcriptome disturbances, at least in mice.

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DNA replication timing in the one-cell zygote. A zygote was pulse-labelled by bromodeoxyuridine (BrdU) for 1 h, immediately fixed by formaldehyde and stained with an anti-BrdU antibody. The pulse-label time intervals from the moment of fertilization are indicated over the pictures. The numbers below the pictures represent ‘BrdU positive zygote’/‘zygote examined’. This result shows that DNA replication starts at 5–6 h after fertilization in both the paternal and maternal pronuclei and terminates at 8–9 h after fertilization. pPN, paternal pronucleus; mPN, maternal pronucleus; PB, polar body.
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RSTB20120353F2: DNA replication timing in the one-cell zygote. A zygote was pulse-labelled by bromodeoxyuridine (BrdU) for 1 h, immediately fixed by formaldehyde and stained with an anti-BrdU antibody. The pulse-label time intervals from the moment of fertilization are indicated over the pictures. The numbers below the pictures represent ‘BrdU positive zygote’/‘zygote examined’. This result shows that DNA replication starts at 5–6 h after fertilization in both the paternal and maternal pronuclei and terminates at 8–9 h after fertilization. pPN, paternal pronucleus; mPN, maternal pronucleus; PB, polar body.

Mentions: At the one-cell stage, 5-methylcytosine (5mC) in the male pronucleus is rapidly converted to 5-hydroxymethylcytosine (5hmC; figure 1b), and this conversion is not observed in the female pronucleus. The Tet1, Tet2 and Tet3 enzymes catalyse the conversion reaction from 5mC to 5hmC. Experimental investigation using both an anti-5hmC antibody and an anti-5mC antibody clearly demonstrated that the rapid disappearance of the 5mC in the male pronucleus is due to the conversion to 5hmC by the Tet3 enzyme [26,27]. Maternal germ-cell-specific Tet3 disruption results in there being no preferential 5mC conversion to 5hmC in the paternal pronucleus. Oocytes lacking the Tet3 enzyme exhibit a severe effect on embryonic development [28]. The conversion reaction in the paternal pronucleus starts at 4–6 h after fertilization, and a marked asymmetry in the male and female pronucleus is clearly evident 8 h after fertilization. The first DNA replication also starts in almost the same period in both pronuclei [29,30]. In an unpublished experimental result, the DNA synthesis started at 5–6 h after fertilization and terminated before 9 h (figure 2; T. Kohda 2012, unpublished data). An ‘active demethylation’ mechanism was reportedly proposed in this process, in which 5mC is converted to thymine by activation-induced cytidine deaminase and then the TG mismatched base pair is restored by a DNA repair mechanism [31]. It is also reported that 5hmC is further oxidized to 5-formylcytosine and 5-carboxylcytosine in the paternal allele by Tet enzymes [32,33]. However, several lines of evidence have suggested that most of the 5hmC in the male pronucleus as well as 5mC in the female pronucleus is subjected to ‘passive demethylation’ [26,27,34] (figure 1c).Figure 2.


Embryo manipulation via assisted reproductive technology and epigenetic asymmetry in mammalian early development.

Kohda T, Ishino F - Philos. Trans. R. Soc. Lond., B, Biol. Sci. (2013)

DNA replication timing in the one-cell zygote. A zygote was pulse-labelled by bromodeoxyuridine (BrdU) for 1 h, immediately fixed by formaldehyde and stained with an anti-BrdU antibody. The pulse-label time intervals from the moment of fertilization are indicated over the pictures. The numbers below the pictures represent ‘BrdU positive zygote’/‘zygote examined’. This result shows that DNA replication starts at 5–6 h after fertilization in both the paternal and maternal pronuclei and terminates at 8–9 h after fertilization. pPN, paternal pronucleus; mPN, maternal pronucleus; PB, polar body.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539368&req=5

RSTB20120353F2: DNA replication timing in the one-cell zygote. A zygote was pulse-labelled by bromodeoxyuridine (BrdU) for 1 h, immediately fixed by formaldehyde and stained with an anti-BrdU antibody. The pulse-label time intervals from the moment of fertilization are indicated over the pictures. The numbers below the pictures represent ‘BrdU positive zygote’/‘zygote examined’. This result shows that DNA replication starts at 5–6 h after fertilization in both the paternal and maternal pronuclei and terminates at 8–9 h after fertilization. pPN, paternal pronucleus; mPN, maternal pronucleus; PB, polar body.
Mentions: At the one-cell stage, 5-methylcytosine (5mC) in the male pronucleus is rapidly converted to 5-hydroxymethylcytosine (5hmC; figure 1b), and this conversion is not observed in the female pronucleus. The Tet1, Tet2 and Tet3 enzymes catalyse the conversion reaction from 5mC to 5hmC. Experimental investigation using both an anti-5hmC antibody and an anti-5mC antibody clearly demonstrated that the rapid disappearance of the 5mC in the male pronucleus is due to the conversion to 5hmC by the Tet3 enzyme [26,27]. Maternal germ-cell-specific Tet3 disruption results in there being no preferential 5mC conversion to 5hmC in the paternal pronucleus. Oocytes lacking the Tet3 enzyme exhibit a severe effect on embryonic development [28]. The conversion reaction in the paternal pronucleus starts at 4–6 h after fertilization, and a marked asymmetry in the male and female pronucleus is clearly evident 8 h after fertilization. The first DNA replication also starts in almost the same period in both pronuclei [29,30]. In an unpublished experimental result, the DNA synthesis started at 5–6 h after fertilization and terminated before 9 h (figure 2; T. Kohda 2012, unpublished data). An ‘active demethylation’ mechanism was reportedly proposed in this process, in which 5mC is converted to thymine by activation-induced cytidine deaminase and then the TG mismatched base pair is restored by a DNA repair mechanism [31]. It is also reported that 5hmC is further oxidized to 5-formylcytosine and 5-carboxylcytosine in the paternal allele by Tet enzymes [32,33]. However, several lines of evidence have suggested that most of the 5hmC in the male pronucleus as well as 5mC in the female pronucleus is subjected to ‘passive demethylation’ [26,27,34] (figure 1c).Figure 2.

Bottom Line: The sperm and egg DNA methylation profiles are very different from each other, and just after fertilization, only the paternally derived genome is subjected to genome-wide hydroxylation of 5-methylcytosine, resulting in an epigenetic asymmetry in parentally derived genomes.Zygotic gene activation from paternally or maternally derived genomes also starts around the two-cell stage, presumably in a different manner in each of them.In particular, we focus on the effects of intracytoplasmic sperm injection that can result in long-lasting transcriptome disturbances, at least in mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Epigenetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.

ABSTRACT
The early stage of mammalian development from fertilization to implantation is a period when global and differential changes in the epigenetic landscape occur in paternally and maternally derived genomes, respectively. The sperm and egg DNA methylation profiles are very different from each other, and just after fertilization, only the paternally derived genome is subjected to genome-wide hydroxylation of 5-methylcytosine, resulting in an epigenetic asymmetry in parentally derived genomes. Although most of these differences are not present by the blastocyst stage, presumably due to passive demethylation, the maintenance of genomic imprinting memory and X chromosome inactivation in this stage are of critical importance for post-implantation development. Zygotic gene activation from paternally or maternally derived genomes also starts around the two-cell stage, presumably in a different manner in each of them. It is during this period that embryo manipulation, including assisted reproductive technology, is normally performed; so it is critically important to determine whether embryo manipulation procedures increase developmental risks by disturbing subsequent gene expression during the embryonic and/or neonatal development stages. In this review, we discuss the effects of various embryo manipulation procedures applied at the fertilization stage in relation to the epigenetic asymmetry in pre-implantation development. In particular, we focus on the effects of intracytoplasmic sperm injection that can result in long-lasting transcriptome disturbances, at least in mice.

Show MeSH