131DIFFERENTIAL DNA METHYLATION CHANGES OF THE REPETITIVE SEQUENCES 
DURING PREIMPLANTATION DEVELOPMENT IN THE MOUSE

DNA demethylation as an epigenetic change is a unique event genome-wide, occurring at preimplantation and germ cell stages during mouse development. The paternal genome after fertilization is demethylated first, referred to active demethylation, followed by demethylation of the maternal genome during preimplantation development in the mouse. To examine methylation changes in the early mouse embryo, methylation states of various genomic regions such as intracisternal A-particle (IAP), early retrotransposon (Etn) and centromeric satellite sequences were determined by means of a bisulfite sequencing method. For methylation analysis, genomic DNA was first isolated from each developmental stage of embryo (about 300 cells in total), respectively, and exposed to 1.9M sodium bisulfite overnight. Targeted DNA sequences were amplified from bisulfite-treated genomic DNAs by PCR, cloned into pGEM T-easy vector and sequenced. Results indicated that IAP sequences maintained high levels of methylation until the morula stage and were demethylated in blastocysts. In contrast to the IAP sequences, methylation states of Etn elements were remarkably erased after fertilization, completely demethylated at the 8-cell stage and then remethylated at the morula stage. Centromeric satellite sequences showed low methylation states throughout all preimplantation stages of embryos, indicating that the satellite sequences are substantially demethylated in both paternal and maternal genomes. The results suggest that differential epifenetic changes among the repetitive sequences may be responsible for peculiar chromatin structure of respective genomic loci and/or may regulate gene expression during preimplantation development in the mouse.

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IGF-2 receptors are first expressed at the 2-cell stage of mouse development

Development ◽

10.1242/dev.111.4.1057 ◽

1991 ◽

Vol 111 (4) ◽

pp. 1057-1060

Author(s):

M.B. Harvey ◽

P.L. Kaye

Keyword(s):

Preimplantation Development ◽

Mouse Development ◽

Cell Stage ◽

Receptor Mrna ◽

Embryonic Genome

A specific IGF-2 receptor antiserum was used to reveal the presence of IGF-2 receptors during preimplantation development of mice. Receptors were present on 2-, 4- and 8-cell embryos, morulae, blastocysts, and on ICMs isolated prior to staining. There was no evidence for receptors on fertilized eggs. These observations confirm reports of the expression of IGF-2 receptor mRNA as early as the 2-cell stage and refine similar observations in blastocysts to confirm expression in both the TE and ICM. A potential auto/paracrine loop is thus one of the first products of activation of the embryonic genome and is expressed constitutively through preimplantation development.

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131 DYNAMICS OF HISTONE H3 METHYLATION AT POSITIONS K4 AND K9 IN MOUSE, RABBIT, AND BOVINE PRE-IMPLANTATION EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab131 ◽

2006 ◽

Vol 18 (2) ◽

pp. 174 ◽

Cited By ~ 1

Author(s):

K. Lepikhov ◽

F. Yang ◽

C. Wrenzycki ◽

V. Zakhartchenko ◽

H. Niemann ◽

...

Keyword(s):

Histone H3 ◽

Distribution Patterns ◽

Blastocyst Stage ◽

Dna Demethylation ◽

Preimplantation Embryos ◽

Staining Procedure ◽

Cell Stage ◽

Paternal Genome ◽

Histone H3 Methylation ◽

Rabbit Embryos

In mammals, upon the penetration of sperm into the oocyte, the paternal genome undergoes dramatic epigenetic changes. Protamin packaging of DNA is replaced by histones that acquire specific modifications. In mouse zygotes, paternal DNA gets rapidly demethylated by an active mechanism. In bovine zygotes the methylation from paternal DNA is erased only partially, and in rabbit zygotes it persists at the initial level. To understand whether these reprogramming differences are also reflected in histone modifications, we examined the dynamic changes of histone H3 methylation at positions K4 and K9 in mouse, bovine, and rabbit zygotes and in preimplantation embryos using an immunofluorescence staining procedure (Lepikhov and Walter 2004 BMC Dev. Biol. 4, 12). In zygotes, maternal chromatin contains both types of histone H3 methylation. After fertilization protamines in sperm are very quickly replaced by histones. After the formation of nucleosomes, histone H3 acquires methylation at position K4 in a stepwise manner: first as mono-methylated form and later as tri-methylated. In the late zygote, both paternal and maternal pronuclei show equal levels of histone H3 methylation at position K4. Regardless of the differences in DNA reprogramming in these 3 species, H3/K9 di-methylation is not detected on paternal genomes and is only associated with maternal genomes. During the subsequent cleavage stages, H3/K9 di-methylation decreases gradually and becomes hardly detectable in 4-cell bovine and rabbit embryos. In mouse embryos, it is detectable through all the stages. Bovine embryos reacquire this type of modification at the 8-16 cell stage, and it remains at the very low levels in rabbit, embryos until the blastocyst stage. In conclusion, mouse, rabbit and bovine zygotes show similar patterns of H3/K4triMe and H3/K9diMe distribution despite the difference in paternal DNA demethylation. The dynamics of H3/K9diMe distribution patterns in cleavage stage embryos from all embryos do not correlate with embryonic genomic activation events.

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Expression of ZO-1 and occludin at mRNA and protein level during preimplantation development of the pig parthenogenetic diploids

Zygote ◽

10.1017/s0967199410000705 ◽

2011 ◽

Vol 20 (2) ◽

pp. 147-158 ◽

Cited By ~ 6

Author(s):

Shangdan Xu ◽

Jibak Lee ◽

Masashi Miyake

Keyword(s):

Western Blotting ◽

Contact Region ◽

Relative Concentration ◽

Preimplantation Development ◽

Preimplantation Embryos ◽

Homogeneous Distribution ◽

Mrna Levels ◽

Cell Stage ◽

Specific Expression ◽

Morula Stage

SummaryExpression of mRNAs and proteins of ZO-1 and occludin was analyzed in pig oocytes and parthenogenetic diploid embryos during preimplantation development using real-time RT-PCR, western blotting and immunocytochemistry. All germinal vesicle (GV) and metaphase (M)II oocytes and preimplantation embryos expressed mRNAs and proteins of ZO-1 and occludin. mRNA levels of both ZO-1 and occludin decreased significantly from GV to MII, but increased at the 2-cell stage followed by temporal decrease during the early and late 4-cell stages. Then, both mRNAs increased after compaction. Relative concentration of zo1α− was highest in 2-cell embryos, while zo1α+ was expressed from the morula stage. Occludin expression greatly increased after the morula stage and was highest in expanded blastocysts. Western blotting analysis showed constant expression of ZO-1α− throughout preimplantation development and limited translation of ZO-1α+ from the blastocysts, and species-specific expression pattern of occludin. Immunocytochemistry analysis revealed homogeneous distribution of ZO-1 and occludin in the cytoplasm with moderately strong fluorescence in the vicinity of the contact region between blastomeres, around the nuclei in the 2-cell to late 4-cell embryos, and clear network localization along the cell-boundary region in embryos after the morula stage. Present results show that major TJ proteins, ZO-1 and occludin are expressed in oocytes and preimplantation embryos, and that ZO-1α+ is transcribed by zygotic gene activation and translated from early blastocysts with prominent increase of occludin at the blastocyst stage.

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Chromosomal position and specific demethylation in enhancer sequences of germ line-transmitted retroviral genomes during mouse development

Molecular and Cellular Biology ◽

10.1128/mcb.5.9.2212-2220.1985 ◽

1985 ◽

Vol 5 (9) ◽

pp. 2212-2220

Author(s):

D Jähner ◽

R Jaenisch

Keyword(s):

Dna Sequences ◽

De Novo ◽

Germ Line ◽

Gene Activation ◽

Dna Demethylation ◽

Proviral Genome ◽

Mouse Development ◽

Cpg Sites ◽

Chromosomal Position ◽

Enhancer Sequences

The methylation pattern of the germ line-transmitted Moloney leukemia proviral genome was analyzed in DNA of sperm, of day-12 and day-17 embryos, and of adult mice from six different Mov substrains. At day 12 of gestation, all 50 testable CpG sites in the individual viral genomes as well as sites in flanking host sequences were highly methylated. Some sites were unmethylated in sperm, indicating de novo methylation of unique DNA sequences during normal mouse development. At subsequent stages of development, specific CpG sites which were localized exclusively in the 5' and 3' enhancer regions of the long terminal repeat became progressively demethylated in all six proviruses. The extent of enhancer demethylation, however, was tissue specific and strongly affected by the chromosomal position of the respective proviral genome. This position-dependent demethylation of enhancer sequences was not accompanied by a similar change within the flanking host sequences, which remained virtually unchanged. Our results indicate that viral enhancer sequences, but not other sequences in the M-MuLV genome, may have an intrinsic ability to interact with cellular proteins, which can perturb the interaction of the methylase with DNA. Demethylation of enhancer sequences is not sufficient for gene expression but may be a necessary event which enables the enhancer to respond to developmental signals which ultimately lead to gene activation.

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Expression profile of protein kinase C isozymes in preimplantation mouse development

Reproduction ◽

10.1530/rep.1.00571 ◽

2005 ◽

Vol 130 (4) ◽

pp. 441-451 ◽

Cited By ~ 13

Author(s):

Hesam Dehghani ◽

Ann C Hahnel

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Mouse Embryo ◽

Mitotic Cell ◽

Preimplantation Development ◽

Mouse Development ◽

Cell Stage ◽

Kinase C ◽

Preimplantation Mouse ◽

The Individual

In the preimplantation mouse embryo, the protein kinase C (PKC) family has been implicated in regulation of egg activation, progression of meiotic and mitotic cell cycles, embryo compaction, and blastulation, but the involvement of the individual isozymes is largely unknown. Here, using semiquantitative immunocytochemistry and confocal microscopy we analyze the relative amount and subcellular distribution of ten isozymes of PKC (α, βI, βII, γ, δ, ε, η, 𝛉, ζ, ι/λ) and a PKC-anchoring protein, receptor for activated C-kinase 1 (RACK1). Our results show that all of these isoforms of PKC are present between the two-cell and blastocyst stages of mouse preimplantation development, and that each has a distinct, dynamic pattern and level of expression. The data suggest that different complements of the isozymes are involved in various steps of preimplantation development, and will serve as a framework for further functional studies of the individual isozymes. In particular, there was a transient increase in the nuclear concentration of several isozymes at the early four-cell stage, suggesting that some of the PKC isozymes might be involved in regulation of nuclear organization and function in the early mouse embryo.

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Genome reprogramming during the first cell cycle in in vitro produced porcine embryos

Czech Journal of Animal Science ◽

10.17221/26/2009-cjas ◽

2010 ◽

Vol 55 (No. 2) ◽

pp. 49-57 ◽

Cited By ~ 2

Author(s):

I. Barnetová ◽

K. Okada

Keyword(s):

Intracytoplasmic Sperm Injection ◽

Methylation Pattern ◽

Dna Demethylation ◽

Embryo Production ◽

Cell Stage ◽

Paternal Genome ◽

Labelling Intensity ◽

Mammalian Embryos ◽

Methylation Patterns

Conflicting data still exist regarding the extent of paternal pronuclear DNA demethylation in one cell-stage mammalian embryos. Demethylation of paternal pronuclear DNA was observed in in vitro produced porcine zygotes, whereas in vitro produced embryos do not show any or only weak paternal genome demethylation. In our experiments, we have used and compared two in vitro techniques commonly used for in vitro embryo production (in vitro fertilization and intracytoplasmic sperm injection) and then we evaluated the extent of labelling in both these groups after 5-methylcytosine (5-MeC) or dimethyl H3/K9 labelling. We have found no differences in the methylation pattern between both those techniques used for the production of embryos. Moreover, we did not detect any demethylation of paternal DNA after 5-MeC labelling at all. Contrary to this, labelling with dimethyl H3/K9 antibodies showed differences in labelling intensity between maternal and paternal genomes in 42% of zygotes after in vitro fertilization and in 44% of zygotes after intracytoplasmic sperm injection. Our results indicate that in vitro matured pig oocytes exhibit rather inconsistent methylation patterns. This inconsistency probably resulted from insufficient cytoplasmic maturation of oocytes and to a lesser extent from the in vitro technique for embryo production.

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Chromosomal position and specific demethylation in enhancer sequences of germ line-transmitted retroviral genomes during mouse development.

Molecular and Cellular Biology ◽

10.1128/mcb.5.9.2212 ◽

1985 ◽

Vol 5 (9) ◽

pp. 2212-2220 ◽

Cited By ~ 38

Author(s):

D Jähner ◽

R Jaenisch

Keyword(s):

Dna Sequences ◽

De Novo ◽

Germ Line ◽

Gene Activation ◽

Dna Demethylation ◽

Proviral Genome ◽

Mouse Development ◽

Cpg Sites ◽

Chromosomal Position ◽

Enhancer Sequences

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Symmetrically dimethylated histone H3R2 promotes global transcription during minor zygotic genome activation in mouse pronuclei

Scientific Reports ◽

10.1038/s41598-021-89334-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kohtaro Morita ◽

Yuki Hatanaka ◽

Shunya Ihashi ◽

Masahide Asano ◽

Kei Miyamoto ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Arginine Methylation ◽

Dna Demethylation ◽

Zygotic Genome Activation ◽

H3k4 Methylation ◽

Cell Stage ◽

Paternal Genome ◽

Genome Activation ◽

Histone Arginine Methylation ◽

Zygotic Genome

AbstractPaternal genome reprogramming, such as protamine–histone exchange and global DNA demethylation, is crucial for the development of fertilised embryos. Previously, our study showed that one of histone arginine methylation, asymmetrically dimethylated histone H3R17 (H3R17me2a), is necessary for epigenetic reprogramming in the mouse paternal genome. However, roles of histone arginine methylation in reprogramming after fertilisation are still poorly understood. Here, we report that H3R2me2s promotes global transcription at the 1-cell stage, referred to as minor zygotic genome activation (ZGA). The inhibition of H3R2me2s by expressing a histone H3.3 mutant H3.3R2A prevented embryonic development from the 2-cell to 4-cell stages and significantly reduced global RNA synthesis and RNA polymerase II (Pol II) activity. Consistent with this result, the expression levels of MuERV-L as minor ZGA transcripts were decreased by forced expression of H3.3R2A. Furthermore, treatment with an inhibitor and co-injection of siRNA to PRMT5 and PRMT7 also resulted in the attenuation of transcriptional activities with reduction of H3R2me2s in the pronuclei of zygotes. Interestingly, impairment of H3K4 methylation by expression of H3.3K4M resulted in a decrease of H3R2me2s in male pronuclei. Our findings suggest that H3R2me2s together with H3K4 methylation is involved in global transcription during minor ZGA in mice.

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Simultaneous deletion of the methylcytosine oxidases Tet1 and Tet3 increases transcriptome variability in early embryogenesis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1510510112 ◽

2015 ◽

Vol 112 (31) ◽

pp. E4236-E4245 ◽

Cited By ~ 48

Author(s):

Jinsuk Kang ◽

Matthias Lienhard ◽

William A. Pastor ◽

Ashu Chawla ◽

Mark Novotny ◽

...

Keyword(s):

Single Cells ◽

Cholesterol Biosynthesis ◽

Early Embryogenesis ◽

Dna Demethylation ◽

Cell Stage ◽

Tet Proteins ◽

Genes Encoding ◽

Variable Gene ◽

Intracellular Pathways ◽

Tet Enzymes

Dioxygenases of the TET (Ten-Eleven Translocation) family produce oxidized methylcytosines, intermediates in DNA demethylation, as well as new epigenetic marks. Here we show data suggesting that TET proteins maintain the consistency of gene transcription. Embryos lacking Tet1 and Tet3 (Tet1/3 DKO) displayed a strong loss of 5-hydroxymethylcytosine (5hmC) and a concurrent increase in 5-methylcytosine (5mC) at the eight-cell stage. Single cells from eight-cell embryos and individual embryonic day 3.5 blastocysts showed unexpectedly variable gene expression compared with controls, and this variability correlated in blastocysts with variably increased 5mC/5hmC in gene bodies and repetitive elements. Despite the variability, genes encoding regulators of cholesterol biosynthesis were reproducibly down-regulated in Tet1/3 DKO blastocysts, resulting in a characteristic phenotype of holoprosencephaly in the few embryos that survived to later stages. Thus, TET enzymes and DNA cytosine modifications could directly or indirectly modulate transcriptional noise, resulting in the selective susceptibility of certain intracellular pathways to regulation by TET proteins.

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Spatiotemporal dynamics of OCT4 protein localization during preimplantation development in mice

Reproduction ◽

10.1530/rep-16-0277 ◽

2016 ◽

Vol 152 (5) ◽

pp. 417-430 ◽

Cited By ~ 10

Author(s):

Atsushi Fukuda ◽

Atsushi Mitani ◽

Toshiyuki Miyashita ◽

Hisato Kobayashi ◽

Akihiro Umezawa ◽

...

Keyword(s):

Nuclear Localization ◽

Splice Variants ◽

Protein Localization ◽

Preimplantation Development ◽

Preimplantation Embryos ◽

Sequencing Analysis ◽

Dependent Manner ◽

Cell Stage ◽

Protein Levels ◽

Oct4 Protein

Spatiotemporal expression of transcription factors is crucial for genomic reprogramming. Pou5f1 (Oct4) is an essential transcription factor for reprogramming. A recent study reported that OCT4A, which is crucial for establishment and maintenance of pluripotent cells, is expressed in oocytes, but maternal OCT4A is dispensable for totipotency induction. Whereas another study reported that OCT4B, which is not related to pluripotency, is predominantly expressed instead of OCT4A during early preimplantation phases in mice. To determine the expression states of OCT4 in murine preimplantation embryos, we conducted in-depth expression and functional analyses. We found that pluripotency-related OCT4 mainly localizes to the cytoplasm in early preimplantation phases, with no major nuclear localization until the 8–16-cell stage despite high expression in both oocytes and early embryos. RNA-sequencing analysis using oocytes and early preimplantation embryos could not identify the splice variants creating alternative forms of OCT4 protein. Forced expression of OCT4 in zygotes by the injection of polyadenylated mRNA clearly showed nuclear localization of OCT4 protein around 3–5-fold greater than physiological levels and impaired developmental competency in a dose-dependent manner. Embryos with modest overexpression of OCT4 could develop to the 16-cell stage; however, more than 50% of the embryos were arrested at this stage, similar to the results for OCT4 depletion. In contrast, extensive overexpression of OCT4 resulted in complete arrest at the 2-cell stage accompanied by downregulation of zygotically activated genes and repetitive elements related to the totipotent state. These results demonstrated that OCT4 protein localization was spatiotemporally altered during preimplantation development, and strict control of Oct4 protein levels was essential for proper totipotential reprogramming.

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