Assessment of 'one-step' versus 'sequential' embryo culture conditions through embryonic genome methylation and hydroxymethylation changes.
In comparison to in vivo development, how do different conditions of in vitro culture ('one step' versus 'sequential medium') impact DNA methylation and hydroxymethylation in preimplantation embryos?



Using rabbit as a model, we show that DNA methylation and hydroxymethylation are both affected by in vitro culture of preimplantation embryos and the effect observed depends on the culture medium used.



Correct regulation of DNA methylation is essential for embryonic development and DNA hydroxymethylation appears more and more to be a key player. Modifications of the environment of early embryos are known to have long term effects on adult phenotypes and health; these probably rely on epigenetic alterations.



The study design we used is both cross sectional (control versus treatment) and longitudinal (time-course). Each individual in vivo experiment used embryos flushed from the donor at the 2-, 4-, 8-, 16- or morula stage. Each stage was analyzed in at least two independent experiments. Each individual in vitro experiment used embryos flushed from donors at the 1-cell stage (19 h post-coïtum) which were then cultured in parallel in the two tested media until the 2-, 4-, 8- 16-cell or morula stages. Each stage was analyzed in at least three independent experiments. In both the in vivo and in vitro experiments, 4-cell stage embryos were always included as an internal reference.



Immunofluorescence with antibodies specific for 5-methylcytosine (5meC) and 5-hydroxymethylcytosine (5hmeC) was used to quantify DNA methylation and hydroxymethylation levels in preimplantation embryos. We assessed the expression of DNA methyltransferases (DNMT), of ten eleven translocation (TET) dioxigenases and of two endogenous retroviral sequences (ERV) using RT-qPCR, since the expression of endogenous retroviral sequences is known to be regulated by DNA methylation. Three repeats were first done for all stages; then three additional repetitions were performed for those stages showing differences or tendencies toward differences between the different conditions in the first round of quantification.



The kinetics of DNA methylation and hydroxymethylation were modified in in vitro cultured embryos, and the observed differences depended on the type of medium used. These differences were statistically significant. In addition, the expression of TET1 and TET2 was significantly reduced in post-embryonic genome activation (EGA) embryos after in vitro culture in both tested conditions. Finally, the expression of two retroviral sequences was analyzed and found to be significantly affected by in vitro culture.



Our study remains mostly descriptive as no direct link can be established between the epigenetic changes observed and the expression changes in both effectors and targets of the studied epigenetic modifications. The results we obtained suggest that gene expression could be affected on a large scale, but this remains to be confirmed.



Our results are in agreement with the literature, showing that DNA methylation is sensitive to in vitro culture. As we observed an effect of both tested culture conditions on the tested epigenetic marks and on gene expression, we cannot conclude which medium is potentially closest to in vivo conditions. However, as the observed effects are different, additional studies may provide more information and potential recommendations for the use of culture media in assisted reproductive technology.



This work was supported by an 'AMP diagnostic prénatal et diagnostic génétique' 2012 grant from the French Agence de la Biomédecine. This study was performed within the framework of ANR LABEX 'REVIVE' (ANR-10-LABX-73). Authors are members of RGB-Net (TD 1101) and Epiconcept (FA 1201) COST actions. The authors declare that there is no competing interest.
UMR BDR, INRA, ENVA, Université Paris Saclay, 78350 Jouy en Josas, France veronique.duranthon@jouy.inra.fr juliette.salvaing@cea.fr.|UMR BDR, INRA, ENVA, Université Paris Saclay, 78350 Jouy en Josas, France.
J
J
Salvaing
UMR BDR, INRA, ENVA, Université Paris Saclay, 78350 Jouy en Josas, France.
N
N
Peynot
UMR BDR, INRA, ENVA, Université Paris Saclay, 78350 Jouy en Josas, France.|Present address: Jigjiga University, Ethiopia.
M N
MN
Bedhane
UMR BDR, INRA, ENVA, Université Paris Saclay, 78350 Jouy en Josas, France.
S
S
Veniel
UMR BDR, INRA, ENVA, Université Paris Saclay, 78350 Jouy en Josas, France.|Present address: Faculté de Médecine, 27 Bd Jean Moulin, 13385 Marseille Cedex C5, France.
E
E
Pellier
UMR BDR, INRA, ENVA, Université Paris Saclay, 78350 Jouy en Josas, France.
C
C
Boulesteix
UMR BDR, INRA, ENVA, Université Paris Saclay, 78350 Jouy en Josas, France.|Present address: INSERM U1208, INRA USC1361 Stem Cell and Brain Research Institute Department of Pluripotent Stem Cells in Mammals, 18 avenue Doyen Lépine, 69675 Bron, France.
N
N
Beaujean
UMR BDR, INRA, ENVA, Université Paris Saclay, 78350 Jouy en Josas, France.
N
N
Daniel
UMR BDR, INRA, ENVA, Université Paris Saclay, 78350 Jouy en Josas, France veronique.duranthon@jouy.inra.fr juliette.salvaing@cea.fr.
V
V
Duranthon
D000818:Animals; D001755:Blastocyst / Q000502:physiology*; D003470:Culture Media; D019175:DNA Methylation; D046149:Embryo Culture Techniques / Q000379:methods*; D047108:Embryonic Development / Q000502:physiology*; D005260:Female; D011247:Pregnancy; D011817:Rabbits
D003470:Culture Media
DNA hydroxymethylation; DNA methylation; DNA methyltransferases (DNMT); embryo culture media; endogenous retrovirus; preimplantation embryo; ten eleven translocation (TET)
Hum Mol Genet. 2015 Dec 15;24(24):7121-31
26423458
Theriogenology. 2000 Oct 15;54(7):1109-16
11131329
J Reprod Fertil. 1999 Sep;117(1):125-33
10645253
Proc Natl Acad Sci U S A. 2011 Mar 1;108(9):3642-7
21321204
Virology. 2011 Mar 15;411(2):273-87
21251689
BMC Dev Biol. 2007 Oct 18;7:116
17949482
Br J Nutr. 2007 Jun;97(6):1036-46
17381976
Physiol Genomics. 2009 Jan 8;36(2):98-113
19001509
Hum Reprod. 2011 Sep;26(9):2576-85
21685140
Dev Cell. 2004 Oct;7(4):597-606
15469847
Biol Reprod. 1988 Sep;39(2):295-307
2902884
BMC Mol Biol. 2008 Jul 28;9:67
18662377
Biol Reprod. 1987 Nov;37(4):775-8
3689846
Nat Commun. 2011;2:241
21407207
Cell Stem Cell. 2011 Feb 4;8(2):200-13
21295276
Fertil Steril. 2014 Sep;102(3):759-766.e9
24998366
Nature. 2011 Apr 21;472(7343):370-4
21471966
Reproduction. 2009 Aug;138(2):289-99
19465487
Hum Reprod Update. 2014 Sep-Oct;20(5):632-40
24795173
Nature. 2014 Jul 31;511(7511):606-10
25079557
Trends Biochem Sci. 2006 Feb;31(2):89-97
16403636
Cell Stem Cell. 2014 Oct 2;15(4):447-58
25220291
Genomics. 2014 Nov;104(5):324-33
25173569
Semin Reprod Med. 2008 Mar;26(2):175-85
18302109
Mol Reprod Dev. 1990 May;26(1):90-100
2189447
Reproduction. 2004 Dec;128(6):703-8
15579587
Epigenetics. 2012 May;7(5):440-6
22419129
Nature. 2012 Mar 28;484(7394):339-44
22456710
Cancer Genet. 2015 May;208(5):167-77
25892122
Semin Reprod Med. 2009 Sep;27(5):417-28
19711252
Hum Reprod Update. 2013 May-Jun;19(3):210-20
23385469
Birth Defects Res C Embryo Today. 2009 Dec;87(4):297-313
19960551
PLoS One. 2012;7(5):e38156
22693592
Cancer Biol Ther. 2008 Aug;7(8):1191-3
18719362
Chromosome Res. 2012 Oct;20(7):837-48
23111490
Biol Reprod. 2000 Jul;63(1):281-93
10859270
Fertil Steril. 2016 Mar;105(3):571-87
26851765
Fertil Steril. 2009 Feb;91(2):305-15
19201275
Mol Cell. 2011 May 20;42(4):451-64
21514197
Fertil Steril. 2013 Mar 1;99(3):632-41
23357453
Nature. 2011 May 19;473(7347):398-402
21460836
Nature. 2010 Aug 26;466(7310):1129-33
20639862
Science. 2011 Oct 14;334(6053):194
21940858
Dev Cell. 2014 Apr 14;29(1):102-11
24735881
Nature. 2000 Feb 3;403(6769):501-2
10676950
Biol Reprod. 2008 Feb;78(2):299-306
17989357
Methods Mol Biol. 2015;1222:181-96
25287347
Cell. 2012 Dec 21;151(7):1417-30
23260135
Exp Cell Res. 1969 May;55(2):261-8
5814377
Nature. 2011 May 19;473(7347):394-7
21552279
Biol Open. 2013 May 10;2(6):613-28
23789112
Hum Reprod Update. 2015 Jul-Aug;21(4):555-7
25904658
Nature. 2011 May 19;473(7347):389-93
21451524
J Reprod Fertil. 1979 Jul;56(2):623-6
480314
Science. 2009 May 15;324(5929):930-5
19372391
Nucleic Acids Res. 2010 Oct;38(19):e181
20685817
Genome Biol. 2007;8(2):R19
17291332
Cell. 2014 May 8;157(4):979-91
24813617
PLoS One. 2012;7(1):e30687
22292019
J Dev Orig Health Dis. 2015 Oct;6(5):377-83
25952250
Clin Epigenetics. 2015 Aug 21;7:87
26300992
Epigenetics. 2011 Aug;6(8):987-93
21725201