Comparison of DNA methylation levels of repetitive loci during bovine development
Kaneda et al. BMC Proceedings
Comparison of DNA methylation levels of repetitive loci during bovine development
Masahiro Kaneda 0
Satoshi Akagi 0
Shinya Watanabe 0
Takashi Nagai 0
0 Reproductive Biology and Technology Research Team, National Institute of Livestock and Grassland Science (NILGS), National Agriculture and Food Research Organization (NARO) , 2 Ikenodai, Tsukuba, Ibaraki 305-0901 , Japan
Background: DNA methylation of cytosine residues in CpG dinucleotide controls gene expression and dramatically changes during development. Its pattern is disrupted in cloned animals suggesting incomplete reprogramming during somatic cell nuclear transfer (the first reprogramming). However, the second reprogramming occurs in the germ cells and epigenetic errors in somatic cells of cloned animals should be erased. To analyze the DNA methylation changes on the spermatogenesis of bulls, we measured DNA methylation levels of three repetitive elements in blastocysts, blood and sperm. Methods: DNA from PBLs (peripheral blood leukocytes), sperm and individual IVF (in vitro fertilized) and parthenogenetic blastocysts was isolated and bisulfite converted. Three repetitive elements; Satellite I, Satellite II and art2 sequences were amplified by PCR with specific pairs of primers. The PCR product was then cut by restriction enzymes and analyzed by agarose gel electrophoresis for determining the DNA methylation levels. Results: Both Satellite I and Satellite II sequences were highly methylated in PBLs, whereas hypo-methylated in sperm and blastocysts. The art2 sequence was half methylated both in PBLs and sperm but less methylated in blastocysts. There was no difference in DNA methylation levels between IVF and parthenogenetic blastocysts. Conclusions: These results suggest that there is a dynamic change of DNA methylation during embryonic development and spermatogenesis in cattle. Satellite I and Satellite II regions are methylated during embryogenesis and then de-methylated during spermatogenesis. However, art2 sequences are not de-methylated during spermatogenesis, suggesting that this region is not reprogrammed during germ cell development. These results show dynamic changes of DNA methylation levels during bovine embryogenesis, especially genome-wide reprogramming in germ cells.
DNA methylation is a major physiological modification
in mammalian genome. Cytosine residues in CpG
dinucleotide pairs are selectively methylated by DNA
methyltransferases and these methylation patterns are
maintained throughout cell division. DNA methylation
alters gene expression patterns in cells and is crucial
for normal mammalian development [1,2]. Usually,
repetitive elements such as centromeric repeats and
transposon sequences are highly methylated and
transcriptionally silenced, called a heterochromatic state.
The methylation patterns are dramatically changed
during embryonic development, from a fertilized egg to a
lot of types of differentiated cells. As DNA methylation
controls gene expression, some sets of genes are
activated/inactivated in particular types of cells during
differentiation. Genomic imprinting, which causes
parentof-origin specific gene expression in mammals and X
chromosome inactivation, which compensates X
chromosome genes dosage between females (XX sex
chromosomes) and males (XY sex chromosomes), are
controlled by DNA methylation and are crucial for
normal mammalian development . Aberrant DNA
methylation patterns were observed in many kinds of
tumour cells .
In mouse germ cells development, it was previously
shown that imprinted genes and repetitive elements
were de-methylated in primordial germ cells  and
then re-methylated during spermatogenesis or oogenesis
. De novo DNA methyltransferases, Dnmt3a and
Dnmt3b and Dnmt3-like protein Dnmt3L are
responsible for establishing sex-specific DNA methylation
patterns both in males and females [7-10]. After
fertilization, there is a passive DNA demethylation in
the preimplantation embryos depending on DNA
replication, however, methylation of imprinted gene escapes
this genome-wide demethylation event. De novo
methylation begins after implantation by Dnmt3a and
Dnmt3b, and these methylation patterns are maintained
throughout development by the maintenance DNA
methyltransferase Dnmt1 [11,12]. However, little is
known about the changes of DNA methylation during
embryogenesis in cattle. Here we report the dynamic
changes of DNA methylation patterns at three repetitive
sequences in bovine blastocysts, somatic cells and
Blood and frozen sperm samples from six Japanese
Black bulls (Wagyu), two Japanese Brown bulls
(Akaushi) and one Holstein bull were obtained and genomic
DNA was extracted by using DNeasy Blood & Tissue
Kit (QIAGEN). Sperm DNA was extracted using the
lysis buffer with 200mM dithiothreitol (Sigma-Aldrich).
DNA from individual blastocysts was isolated as
described previously . IVF and PA embryos were
produced by a standard method .
DNA methylation analysis
Genomic DNA was bisulfite converted by EpiTect
Bisulfite Kits (QIAGEN) according to the manufacturer’s
instructions. Three repetitive elements were amplified
with specific pairs of primers previously described .
The amplified PCR products were then cut by
restriction enzymes (Satellite I by AciI, Satellite II by AccII and
art2 by TaqI). After digestion, each size of the digested
PCR fragments was isolated by 2% agarose gel
We analyzed genomic DNA methylation patterns to
monitor the changes of epigenetic patterns during
bovine embryogenesis and spermatogenesis. We chosen
three repetitive regions; Satellite I, Satellite II and art2
sequences. Satellite sequences are repetitive sequences
at the peri-/centromeric regions of the chromosomes,
whereas art2 sequences are Alu-like short interspersed
nuclear elements (SINEs). First, we analyzed genomic
DNA of bull blood (peripheral blood leukocytes, PBLs)
and sperm for methylation status of the same regions.
We found a large difference in methylation status using
restriction enzyme analysis (Figure 1A-C). Satellite I
sequences were highly methylated in PBLs (almost PCR
fragments were cut by AciI), whereas hypo-methylated
in sperm (almost PCR fragments were not cut by AciI)
(Figure 1A). Satellite II sequences were also
hypermethylated in PBLs but hypo-methylated in sperm
(Figure 1B). However, there were no differences in art2
sequence methylation levels between PBLs and sperm
(Figure 1C). These results clearly indicated that both
Satellite I and Satellite II sequences, which are located
on the centromeric heterochromatic regions, were
demethylated during spermatogenesis, whereas art2
sequences, which are located on euchromatic regions,
were not methylated/de-methylated during
spermatogenesis. Of nine bulls analyzed, there were no
differences in DNA methylation patterns of three repetitive
elements among individuals and breeds (bulls #1 and
#6-9 are Japanese Black, bulls #3-5 are Japanese Brown
and Bull #2 is Holstein).
We also analyzed DNA methylation levels in
individual blastocysts; 10 in vitro fertilized (IVF) and 10
parthenogenetically activated (PA) embryos. Both
Satellite I and Satellite II regions were hypo-methylated,
whereas art2 sequences were moderately methylated in
IVF and PA blastocysts. There were no differences in
DNA methylation patterns between IVF and PA
embryos, however, some blastocysts showed more
methylated patterns (less cut by restriction enzymes)
compared to others (Figure 1A and 1B).
This study shows the DNA methylation changes of
repetitive elements during bovine development. The
methylation pattern differences of imprinted genes IGF2 and
SNRPN in bovine oocytes and sperm have been reported
[15,16] and the methylation levels of repetitive elements in
bovine blastocysts, especially embryos produced by
somatic cell nuclear transfer (SCNT) technology showing
high levels of DNA methylation, have also been described
[14,17]. However, the DNA methylation status of
repetitive elements in sperm DNA has not been well
understood. We found that two satellite sequences on the
centromeric regions of chromosomes, Satellite I and
Satellite II sequences, were highly methylated in PBLs but
hypo-methylated in sperm. As these regions were not
methylated at blastocyst stages, it is suggested that they
are methylated after implantation, and then de-methylated
during spermatogenesis. In contrast, art2 sequences were
moderately methylated in blastocysts but more methylated
Figure 1 DNA methylation analysis in PBLs, sperm and blastocysts. Bulls #1-#9 shows each number of analyzed bulls. Uncut band indicates
the PCR product not cut by restriction enzymes. (A) Satellite I regions were cut by AciI. (B) Satellite II regions were cut by AccII. (C) art2
sequences were cut by TaqI.
both in PBLs and sperm, suggesting this region is
methylated after implantation but not de-methylated during
spermatogenesis. There were no differences between IVF
and PA blastocysts, suggesting that these repetitive
elements are not methylated by a parent-of-origin specific
manner. We analyzed total nine bulls with three different
breeds; six Japanese Black, two Japanese Brown and one
Holstein, however, there were no difference in DNA
methylation patterns among individuals. These results
suggest that in adults DNA methylation patterns are firmly
maintained during embryogenesis and uniformly
reprogrammed during spermatogenesis. In contrast, we
observed differences of DNA methylation patterns among
the individual IVF and PA blastocysts. This could be
explained that the variation itself confers the
developmental competence of embryos because SCNT embryos with
high DNA methylation levels mostly die in utero and even
IVF embryos, only half of them transferred to the uterus
develop to term.
In SCNT embryos, Satellite I, Satellite II regions and
art2 sequences are hyper-methylated compared to IVF
embryos . As donor cells also have high DNA
methylation levels, it is suggested that the first
reprogramming step (transfer donor nucleus to the
enucleated oocyte) is not sufficient to fully reprogram the
donor genome. SCNT technology has been developed to
rewind the differentiation mechanism, however, the
efficiency of this artificial reprogramming is quite low so
that still now, more than ten years has past since the
first cloned sheep Dolly was born, the success rate of
SCNT is still less than 5-10% in cattle and other species.
This incomplete reprogramming in SCNT and the
resulting alternation of DNA methylation and gene
expression were described . However, it is
hypothesized that the epigenetic errors that were not corrected
during the first reprogramming step are erased and then
properly reprogrammed (the second reprogramming
step) during germ cell development . Therefore,
offspring from cloned animals do not show any
abnormalities observed in cloned animals themselves. In fact, the
obese phenotype frequently observed in cloned mice
does not transmitted to the next generation . In
cattle, there is no remarkable difference in health status
and food products among non-cloned, cloned cattle
developed to adulthood and their offspring [21-23]. By
applying this study for cloned cattle, it will be possible
to prove proper epigenetic reprogramming during
cloned cattle gametogenesis and thus contribute to the
normality of cloned cattle offspring.
We thank Kumamoto Prefectural Agriculture Research Center and Ibaraki
Prefectural Livestock Research Center for providing blood and sperm
samples from Japanese Brown and Japanese Black bulls.
This article has been published as part of BMC Proceedings Volume 5
Supplement 4, 2011: Proceedings of the International Symposium on Animal
Genomics for Animal Health (AGAH 2010). The full contents of the
supplement are available online at
MK carried out DNA extraction, DNA methylation analysis and analyzed the
data. SA made IVF and PA embryos. SW provided blood and sperm samples
from bulls. TN participated in the design of the study and contributed to
discussion of the results and revision of the paper. All authors read and
approved the final manuscript.
1. Li E : Chromatin modification and epigenetic reprogramming in mammalian development . Nature Reviews Genetics 2002 , 3 : 662 - 673 .
2. Reik W : Stability and flexibility of epigenetic gene regulation in mammalian development . Nature 2007 , 447 : 425 - 432 .
3. Miyoshi N , Barton SC , Kaneda M , Hajkova P , Surani MA : The continuing quest to comprehend genomic imprinting . Cytogenet Genome Res 2006 , 113 : 6 - 11 .
4. Jones PA , Baylin SB : The epigenomics of cancer . Cell 2007 , 128 : 683 - 692 .
5. Hajkova P , Erhardt S , Lane N , Haaf T , El-Maarri O , Reik W , Walter J , Surani MA : Epigenetic reprogramming in mouse primordial germ cells . Mechanisms of development 2002 , 117 : 15 - 23 .
6. Lane N , Dean W , Erhardt S , Hajkova P , Surani A , Walter J , Reik W : Resistance of IAPs to methylation reprogramming may provide a mechanism for epigenetic inheritance in the mouse . Genesis 2003 , 35 : 88 - 93 .
7. Bourc'his D , Xu GL , Lin CS , Bollman B , Bestor TH : Dnmt3L and the establishment of maternal genomic imprints . Science 2001 , 294 : 2536 .
8. Kaneda M , Okano M , Hata K , Sado T , Tsujimoto N , Li E , Sasaki H : Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting . Nature 2004 , 429 : 900 - 903 .
9. Bourc'his D , Bestor TH : Meiotic catastrophe and retrotransposon reactivation in male germ cells lacking Dnmt3L . Nature 2004 , 431 : 96 - 99 .
10. Kato Y , Kaneda M , Hata K , Kumaki K , Hisano M , Kohara Y , Okano M , Li E , Nozaki M , Sasaki H : Role of the Dnmt3 family in de novo methylation of imprinted and repetitive sequences during male germ cell development in the mouse . Hum Mol Genet 2007 , 16 : 2272 .
11. Okano M , Bell DW , Haber DA , Li E : DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development . Cell 1999 , 99 : 247 - 258 .
12. Hirasawa R , Chiba H , Kaneda M , Tajima S , Li E , Jaenisch R , Sasaki H : Maternal and zygotic Dnmt1 are necessary and sufficient for the maintenance of DNA methylation imprints during preimplantation development . Genes Dev 2008 , 22 : 1607 - 1616 .
13. Akagi S , Hosoe M , Matsukawa K , Ichikawa A , Tanikawa T , Takahashi S : Culture of Bovine Embryos on a Polydimethylsiloxane (PDMS) Microwell Plate . J Reprod Dev 2010 , 56 : 475 - 459 .
14. Kang YK , Koo DB , Park JS , Choi YH , Chung AS , Lee KK , Han YM : Aberrant methylation of donor genome in cloned bovine embryos . Nature genetics 2001 , 28 : 173 - 177 .
15. Gebert C , Wrenzycki C , Herrmann D , Gröger D , Reinhardt R , Hajkova P , Lucas-Hahn A , Carnwath J , Lehrach H , Niemann H : The bovine IGF2 gene is differentially methylated in oocyte and sperm DNA . Genomics 2006 , 88 : 222 - 229 .
16. Lucifero D , Suzuki J , Bordignon V , Martel J , Vigneault C , Therrien J , Filion F , Smith LC , Trasler JM : Bovine SNRPN methylation imprint in oocytes and day 17 in vitro-produced and somatic cell nuclear transfer embryos . Biol Reprod 2006 , 75 : 531 - 538 .
17. Kang YK , Lee HJ , Shim JJ , Yeo S , Kim SH , Koo DB , Lee KK , Beyhan Z , First NL , Han YM : Varied patterns of DNA methylation change between different satellite regions in bovine preimplantation development . Mol Reprod Dev 2005 , 71 : 29 - 35 .
18. Dean W , Santos F , Stojkovic M , Zakhartchenko V , Walter J , Wolf E , Reik W : Conservation of methylation reprogramming in mammalian development: aberrant reprogramming in cloned embryos . Proc Natl Acad Sci U S A 2001 , 98 : 13734 .
19. Fulka J , Miyashita N , Nagai T , Ogura A : Do cloned mammals skip a reprogramming step ? Nat Biotechnol 2004 , 22 : 25 - 26 .
20. Tamashiro KLK , Wakayama T , Akutsu H , Yamazaki Y , Lachey JL , Wortman MD , Seeley RJ , D'Alessio DA , Woods SC , Yanagimachi R : Cloned mice have an obese phenotype not transmitted to their offspring . Nature Medicine 2002 , 8 : 262 - 267 .
21. Shiga K , Umeki H , Shimura H , Fujita T , Watanabe S , Nagai T : Growth and fertility of bulls cloned from the somatic cells of an aged and infertile bull . Theriogenology 2005 , 64 : 334 - 343 .
22. Watanabe S , Nagai T : Health status and productive performance of somatic cell cloned cattle and their offspring produced in Japan . J Reprod Dev 2008 , 54 : 6 - 1 .
23. Watanabe S , Nagai T : Death losses due to stillbirth, neonatal death and diseases in cloned cattle derived from somatic cell nuclear transfer and their progeny: a result of nationwide survey in Japan . Anim Sci J 2009 , 80 : 233 - 238 .