DNA methylation and hydroxymethylation in early rabbit embryos: Consequences of in vitro culture. Bedhane Mohammed

Transcription

1 European Master in Animal Breeding and Geneti cs (EM-ABG) Master Scie ces et Tech ologies du Viva t et de l E viro e e t Spécialité : Génétique Animale, Génome et Diversité DNA methylation and hydroxymethylation in early rabbit embryos: Consequences of in vitro culture Bedhane Mohammed Supervisors: Salvaing Juliette (INRA) Andersson Göran (SLU) June

2 Abstract During the first developmental stages, the genome of the embryo is transcriptionally silent and developmental changes are under the control of maternally inherited factors. Embryonic genome activation (EGA) takes place at later stages (8/16-cell stage in rabbit) and involves epigenetic modifications. DNA methylation at CpG dinucleotides is an epigenetic mark. CpG methylation is depleted at the early stages and reinstated at the blastocyst stage. Recent findings have shown that demethylation involves the oxidation of methylated DNA into hydroxymethylated DNA. However the role of hydroxymethylation can probably not be restricted to an intermediate in DNA demethylation. Indeed, hydroxymethylation seems involved in gene activation and maintenance of pluripotency, and could therefore be important for EGA. Several studies have suggested that in vitro conditions can have a negative impact on epigenetic reprogramming. Therefore, our aim was to investigate the impact of two culture media on methylation and hydroxymethylation in rabbit embryos. To quantify methylated and hydroxymethylated DNA, we implemented an immunofluorescence detection protocol on rabbit embryos cultured in those media until different developmental stages. Our results show that the dynamics of methylation and hydroxymethylation are different between the two culture media conditions. Further investigation is needed to compare the in vitro cultured embryos to in vivo developed ones. Key words: in vitro culture, DNA methylation, hydroxymethylation, rabbit embryo, Assisted Reproductive Technologies 2

3 Summary After the formation of the zygote, embryos undergo a series of cell divisions to ultimately become a blastocyst ready to implant in the uterus. During the first developmental stages, the genome of the embryo is transcriptionally silent and the developmental changes are under control of maternally inherited factors (RNA and proteins). Embryonic genome activation (EGA) is initiated at later stages depending on the species. In rabbit, the major activation phase takes place between the 8-cell and 16-cell stages. It necessitates important reprogramming of epigenetic marks, such as histone modifications or DNA methylation. DNA methylation is an important mark for embryonic development. After fertilization, the genome is demethylated and remethylation occurs only at the blastocyst stage. Recent findings have shown that demethylation is achieved by oxidation of the methylated DNA into hydroxymethylated DNA. However, the role of hydroxymethylation can probably not be restricted to an intermediate product of demethylation. Recent studies have shown that hydroxymethylation could be involved in gene activation, and could therefore play a role in EGA. Several studies have shown that in vitro conditions can have an impact on epigenetic reprogramming; in particular a study from my host laboratory has shown an effect of in vitro culture on DNA methylation. Those modifications that occur during the earliest developmental stages can have an effect that is only observed much later, including after birth or in the adult. This is a particularly sensitive topic as in vitro culture is commonly used in human Assisted Reproductive Technologies (ART), and there are very little data allowing to chose the optimal culture media. Thus, 5-methylcytosine (5MeC) and 5- hydroxymethylcytosine (5hMeC) appeared as interesting candidates to compare the effect of two different culture media conditions on epigenetic reprogramming. We used rabbit as a model because the metabolism and timing of EGA in this species is closer to human embryos than what is observed in mouse. We chose two media that are commonly used for ART, one single-step medium, which allows development from zygote to blastocyst, and one sequential medium, which needs to be changed at the time of EGA. Embryos were fixed at different developmental cell stages: 2, 4, 8, and 16-cell stages. In order to quantify the level of methylated and hydroxymethylated DNA in the nuclei, we implemented an immunofluorescence-based detection protocol. Finally, the methylated and hydroxymethylated DNA were quantified using an appropriate procedure developed in the host laboratory. Our result shows that the dynamics of 5MeC and 5hMeC are different between the two culture media. In the sequential one, methylation increases between 4-cell and 8-cell stages while there is no significant change in hydroxymethylation between 2-cell and 16-cell stages. In the single-step one, hydroxymethylation decreases until the 8-cell stage and increases afterwards, while no change is observed in methylation between 4-cell and 8-cell stages. Further investigations are needed to determine whether one of the culture conditions is closer to the in vivo situation, which could give an indication of which medium is more appropriate for the use in ART. 3

4 Acknowledgements This master thesis project has been completed with the help of many staffs from the institute of INRA and AgroparisTec. First of all I want to give my sincere thanks to my major supervisor Dr. Juliette Salvaing for her unlimited hospitality, support at every step, teaching, guidance, patience, and editing the report with very constructive comments. I was lucky and happy during all my stay with her. Second, I want to thank my second superviso Professor Karl Göran Andersson at Swedish university of agricultural science in Sweden. We are thankful to the Agence de la biomédecine for funding this project. I gratified to MIMA2 platform for accessing of the microscope facility. I would like to thank all BDR young team and other connected staffs: Nathalie Beaujean, Véronique Duranthon, Pierre Adenot, Nathalie Daniel, Nathalie Peynot, Linda Maulny, Eugénie Canon, Claire Boulesteix, Tiphaine Aguirre-Lavin, Amélie Bonnet-Garnier, Martine Chebrout, Renaud Fleurot, and to all students, workers in the secretary office and all others in that beautiful building. Finally, my thanks goes to Etienne Verrier and Gregoire Leroy (APT), Birgitta Malmfors (SLU), Dieuwertje Lont and Britt de Klerk (WU), as well as to my sponsorship EM-ABG program/erasmus mundus and to my family and all friends. 4

5 List of abbreviations 5hMeC 5MeC 5-hydroxymethylcytosine 5-methylcytosine ANOVA Analysis of variance ART BDR BSA DNMT EGA Assisted reproduction technology Biologie Du Développement et Reproduction Bovine Serum Albumin DNA methyltransferase Embryonic genome activation EthD-2 Ethidium Homodimer 2 EXP HAT HDAC hpc HSA ICM INRA Lab PBS PFA SAS TE TET Experiment Histone acetyltransferase Histone deacetylase hour post coitum Human serum albumin Inner Cell Mass Institut National de la Recherche Agronomique Laboratory Phosphate Buffer Solution Paraformaldehyde Statistical analysis system Trophectoderm Ten eleven translocation 5

6 Table of Contents Abstract... 2 Summary... 3 Acknowledgements... 4 List of abbreviations Introduction Early embryo development in mammals Description Embryonic genome activation (EGA) First differentiations Chromatin structure Histones Euchromatin and heterochromatin Epigenetic marks Histone modifications DNA methylation and hydroxymethylation Genomic distribution of 5hMeC Embryo culturing media Materials and Methods Ethics statement Experimental animals Embryo collection and culture The experimental design for embryo development Immunofluorescence

7 2.6. Protocol control Imaging and analysis Statistical analysis Results Dynamics of 5-hydroxymethylcytosine in rabbit embryos Dynamics of 5-methylcytosine in rabbit embryos Discussion Discussion Methylation and hydroxymethylation dynamics up to the 8-cell stage DNA hydroxymethylation and Embryonic Genome Activation (EGA) Effect of in vitro culture Perspectives References Annexes Laboratory Protocol Laboratory book (Lab book) Data recording Format with sample of quantified signal(5hmec) Composition of Global (LifeGlobal) medium: designed formulation to meet all nutritional needs from Day 1 to blastocyst

8 Figure 1: Pre-implantation Development of rabbit embryos. Before compaction, embryos develop in the oviduct environment, while and the end of pre-implantation development takes place in the mammalian uterus (Johnson, 1988). The time line is indicated in hours post-coitum (hpc). 8

9 1. Introduction In this introduction I will provide an overview of mammalian embryonic development and epigenetic marks in the context of embryonic development. I will also introduce in vitro culturing media currently used in assisted reproduction technologies (ART) and the questions the use of these media raise. The project was funded by the Agence de la Biomédecine and implemented by INRA-BDR at Jouy en Josas, France Early embryo development in mammals Description The meeting of the oocyte and sperm, leading to fertilization, takes place in the oviduct. At the 1- cell stage (also called zygote); the two pronuclei (the nucleus of the sperm and egg cell after fertilization) do not fuse and remain separate until the 2-cell stage. In all studied mammalian species, the first developmental stages are characterized by a series of reductive mitotic divisions called cleavages (Figure 1), which remain relatively synchronous until the 8-cell or 16-cell stage depending on the species and become asynchronous at later stages. Compaction is the first event of morphogenic and cellular differentiation (Johnson, 1988). Once the compaction has occurred the embryo begins the process of cavitationresulting in the formation of a blastocyst, which can implant into the uterus. During this developmental period (pre-implantation development) the total size of the embryo does not change but there is a reduction of the size of the blastomeres, allowing to restore a cytoplasmic to nuclear ratio similar to that observed in somatic cells. (Reima et al., 1993; Johnson, 1988) Embryonic genome activation (EGA) During the first stages following fertilization, the genome of the embryo is transcriptionally silent and developmental changes are under post-transcriptional maternal control, i.e. they rely on changes in the translation of mrnas synthesized during oocyte growth, and/or posttranslational protein modifications. The onset of transcription specific to the embryo, referred to as embryonic genome activation (EGA), is initiated later during development at various 9

10 pre-implantation stages depending on species. Embryonic genome activation is established in two phases: minor and major EGA. The minor one shows only a reduced transcriptional activity and does not require any specific transcription factors while the major phase is characterized by rapidly increasing transcription. During the major genome activation, new RNA/proteins are synthesized; those are essential for further embryonic development. (Jeanblanc et. al., 2008; Sonehara et. al., 2008). In rabbit and bovine the major activation takes place between 8-cell and 16-cell stages but in mouse and rat the major activation takes place at the end of the 2-cell stage (Kanka, 2003) First differentiations The first cell fate decisions are made before the occurrence of implantation, at the blastocyst stage. They restrict the cell totipotency which leads to the formation of two different lineages: inner cell mass (ICM) and Trophectoderm (TE). The special feature of the ICM is its pluripotency while the TE cells will differentiate into extraembryonic tissues, in particular the placenta (Johson, 2011; Rossant, 2011) Chromatin structure Chromatin is the complex formed by DNA and histone proteins. It is found only in eukaryotic cells and plays a role to package the DNA into a smaller volume to fit in the cell, to strengthen the DNA (formation of chromosome) to allow mitosis, to prevent DNA damage, and to control DNA replication and gene expression (Cooper, 2000) Histones Histones are highly basic proteins that package and order the DNA into structural units called nucleosomes. Nucleosome is the repeating subunits of chromatin, consisting of a DNA chain coiled around a core of histones. So far five histone families have been identified: H1, H2A, H2B, H3 and H4. These histones are divided broadly between linker histone (H1) and core histone super family (H2A, H2B, H3, H4). Core histones make up the nucleosome and the linker sits on top of the structure and keeps in place the DNA that has wrapped around the nucleosome. Each nucleosome is separated by bp of linker DNA, and the resulting nucleosomal array constitutes a chromatin fiber of 10 nm in diameter which is called beads-on a-string. 10

11 Figure 2: Position of heterochromatin on a mitotic chromosome 11

12 Histones undergo modification in transcriptional regulation including mono-methylation, dimethylation, tri-methylation and acetylation (Brown 2007) Euchromatin and heterochromatin Chromatin's overall structure depends on the stage of the cell cycle. The metaphase chromosomes represent the most compact form and are formed only during nuclear division. On the other hand, during interphase, the chromatin is looser to allow transcription and DNA replication (Bannister and Kouzarides, 2011; Cooper, 2000). Chromatin can be divided in two categories depending on its level of compaction: heterochromatin is tightly packed, while euchromatin is less compacted. Two types of heterochromatin are distinguished: constitutive heterochromatin represents DNA that contains repeated sequences and is maintained in a compact organization in all cell types. This includes pericentromeric, centromeric and telomeric regions of the chromosome (Figure 2). Unlike constitutive heterochromatin, facultative heterochromatin is not a permanent feature of all cells. It contains genes that are inactive in some cells or at some periods of the cell cycle. In general the organization of heterochromatin is so compact that proteins involved in genome expression cannot access this type of DNA (Brown, 2007) Epigenetic marks Histone modifications Histone proteins can undergo various types of modifications such as acetylation, methylation, and phosphorylation. These modifications will affect the interactions between DNA and histones, and thus the level of chromatin compaction, as well as the binding of transcription factors and other regulatory proteins, thus leading to modifications of gene expression (Brown, 2007). The histones can be acetylated on lysine residues in the N-terminal tails. Histone acetylation and deacetylation reactions are catalyzed by enzymes with histone acetyltransferase (HAT) or histone deacetylase (HDAC) activity. The modification associated with acetylation influence chromatin structure by neutralizing the lysine s positive charge resulting in a weakening of the interaction between histone and DNA. Histone methylation occurs on the side chains of lysines and arginines while phosphorylation takes place on serine, threonine and tyrosine residues. 12

13 Figure 3: cytosine, 5-methylcytosine and 5-hydroxymethylcytosine. The methyl group is added to the cytosine at the carbon five position. This is catalyzed by DNA methyltransferases (DNMT); then 5-methylcytosine can be oxidized to 5-hydroxymethylcytosine by ten eleven translocation (TET) proteins. 13

14 Histone methyltransferases catalyze site specific methylation of histone proteins, which, depending on the site, can be involved in either repression or activation of genes. Extensively studied histone methylation sites include H3K4, H3K9, H3K27 H3K36, H3K79, H4K20 which are involved in transcriptional activation, DNA repair, cell cycle regulation and thus play crucial role in growth and development (Greer and Shi, 2012). For example, tri-methylation on lysine-9 of histone H3 forms a biding site for HP1 (Heterochromatin Protein 1), which induces chromatin compaction and silences gene expression: this is the hallmark of constitutive pericentric heterochromatin. Phosphorylation of H3 and the linker histone H1 is associated with the formation of metaphase chromosomes and is involved in controlling the cell cycle (Brown, 2007). In the embryo, H3K27me3 asymmetry was observed on developmentally regulated promoters in the inner cell mass and trophectoderm compartments of mouse blastocyst using ChIP protocol (Dahl et al., 2010) DNA methylation and hydroxymethylation Methylation of mammalian DNA at CpG dinucleotides has long been recognized to play a major role in different cellular functions as development or control of gene expression and it associates with transcriptional repression. It plays a major role in genome activation, X-chromosome inactivation and differentiation that appears to be essential for early embryo development (Corry et al., 2009). The absence of DNA methyltransferases (DNMT), the enzymes, which catalyze this modification, leads to embryonic lethality (Brown, 2007). However, our current study focused only on the dynamics and kinetics of the DNA methylation during pre-implantation embryonic development and this may show the way to understand its biological role and connection with other newly identified epigenetic modifications. DNA methylation involves the addition of a methyl group to the position 5 of the cytosine pyrimidine ring within the CpG dinucleotide motifs in mammals (Figure 3). CpG islands are small regions of the DNA in which the CpG dinucleotide frequency is higher than it would normally be expected. CpG islands are normally not methylated, however, if a CpG island within a promoter becomes methylated the gene associated with the promoter is permanently silenced, and this silencing can be transmitted through mitosis. This means that CpG island methylation is an epigenetic means of inheritance (Brown, 2007). 14

15 So far two types of DNA methylation activities have been distinguished. The first is maintenance methylation, which is dependent on genome replication and ensures that the two daughter cells DNA strands retain the methylation pattern of the parent molecule. The second is de novo methylation, which is independent from DNA replication and adds methyl groups at new positions of the genome (Brown, 2007; Morgan et al., 2005). The enzyme responsible for maintenance methylation activity is called DNA methyltransferase 1 (DNMT1). DNMT3a and DNMT3b are the de novo methyltransferases (Brown, 2007). According to the study of Aapola et al. (2000) their activity increases in the presence of DNMT3L. The opposite process is called DNA demethylation. It can be achieved passively by the failure of the maintenance methylation during DNA synthesis (Brown, 2007), leading to dilution of DNA methylation over the cell cycles. The pathway responsible for active DNA methylation has long remained elusive but the recent discovery of 5-hydroxymethylcytosine (5hMeC) (Tahiliani et al., 2009; Ito et al., 2010) has led to new advances in this research area. The oxidation of 5-methylcytosine (5MeC) results in the formation of 5hMeC. The conversion of 5MeC to 5hMeC is mediated by proteins of the ten eleven translocation (TET) family (Tahiliani et al., 2009). 5hMeC can be further oxidized by TET proteins to 5-carboxylcytosine (5caC) and 5-formylcytosine (5fC) (Ito et al., 2011). In addition, the report of He et al. (2011) has further revealed that oxidation of 5MeC by TET proteins followed by thymine-dna glycosylases (TDG) mediated base excision of 5caC constitutes a pathway for active DNA demethylation. The existence of 5hMeC in the genome requires the pre-existence of 5MeC and it has been confirmed by triple knock-out of DNMT1, DNMT3a and DNMT3b in mouse embryonic stem cells (ESCs) (Ficz et al., 2011). Therefore, 5hMeC is considered to have a direct role in DNA demethylation. In mouse embryos, genome-wide demethylation takes place in the paternal genome (pronucleus) few hours after fertilization and is an active mechanism (Mayer et al., 2000). On the other hand, demethylation of the maternal genome is passive (Rougier et al., 1998) and continues until the morula stage. De novo methylation occurs only at the blastocyst stage and is more important in the inner cell mass (ICM) than in the trophectoderm (TE) (Dean et al., 2001). In rabbit embryos, a first qualitative analysis from Yang et al. (2007) described an absence of demethylation during cleavages and a very slight methylation in ICM but not in trophectoderm cells. However, the quantitative analysis of DNA methylation dynamics in rabbit embryos realized in my host 15

16 laboratory has shown that there is a gradual demethylation of in vivo developed and in vitro cultured embryos during the successive cell cleavages (Reis e Silva et al., 2012). The report also demonstrated that embryos developed in vivo showed a significant decrease in methylation from the 4-cell to the 16-cell stage. In contrast, embryos cultured in vitro showed an earlier decrease of DNA methylation from the 2-cell stage onward until the 8-cell stage. The report of Iqbal et al. (2011) showed that the paternal pronucleus of mouse embryos contains considerable amounts of 5hMeC and low amounts of 5MeC while it is the opposite in the maternal pronucleus. Then 5hMeC remains present into mitotic one-cell, two-cell, and later stages in mouse embryos. Wossidlo et al. (2011) furthermore revealed that 5hMeC could also be detected in bovine and rabbit zygotes. The report also verified that during zygotic development, 5hMeC is accumulated in the paternal pronucleus along with a reduction of 5MeC. Importantly, a previous research report in my host laboratory showed that co-staining with antibodies directed against 5MeC and 5hMec, as was used in the studies cited above, leads to a diminution of the weaker staining, which is probably due to antibody competition (Salvaing et al., 2012). Therefore, in the current study we used only simple staining. There seem to be several ways by which 5hMeC can be removed from the genome. A report from Inoue and Zhang (2011) has shown that there is a replication dependent loss of 5hMeC from the 4-cell stage until the blastocyst. Recently additional pathways have been proposed for the elimination of 5hMeC. Further oxidation followed by the action of DNA glycosylases and Base Excision Repair (BER) is a possible pathway that may be involved in the removal of 5hMeC (Guo et al., 2011) : indeed a recent report has shown that 5fC and 5caC, produced by further oxidation of 5hMeC, are found in mouse embryos (Inoue et al., 2011). Finally, an in vitro study has suggested a surprising role of de novo DNA methyltransferases DNMT3a and DNMT3b in the direct conversion of 5hMeC to unmodified cytosine under oxidative conditions (Chen et al., 2013). So far, considerable advancements have been done on the elimination mechanism of 5hMeC; however, up to date how 5hMeC is maintained during DNA replication is still unknown. 16

17 Genomic distribution of 5hMeC Extensive studies have been done to determine the genomic distribution of 5hMeC in both human and mouse ESCs and brain tissues. The relative abundance of 5hMeC is high in ESCs and brain tissues (Jin et al., 2011). The different affinity-based (antibodies and chemicals labeling) methods explained that 5hMeC is highly clustered in the genome (Yu et al., 2012). According to the study by Williams et al. (2011) 5hMeC is enriched in gene-rich euchromatic regions, particularly at transcription start sites (TSSs), promoters, and exons. In addition, 5hMeC is preferentially located at genomic regions with moderate CpG density. The report by Wu et al. (2011) has confirmed that 5hMeC is relatively enriched in the gene bodies of actively transcribed genes Embryo culturing media A review article by Miller (2004) indicated that in 1882, Ringer designed a tissue culture media from a simple salt solution based on the constituents of blood serum for the in vitro study of the beating frog heart. The solution was composed of sodium chloride, potassium chloride, calcium chloride and a low concentration of sodium bicarbonate. The design of chemically defined media accelerated in the 1940s. During this time, the first culture media were designed for rabbit embryos (Review, Biggers 1987, cited by Zander-Fox and Lane, 2012). Later on, in 1949 mouse embryos were grown in culture media from the 8-cell stage to blastocyst (Zander-Fox and Lane, 2012). During the 1960s and 1970s there were many detailed studies trying to understand more about basic embryo physiological requirement at each developmental stage (Orginal, Whittengham and Biggers, 1976, cited by Zander-Fox and Lane, 2012). In 1978, Louise Joy Brown was the first human born after conception by in vitro fertilization. This opened new possibilities in human embryo culture and assisted reproductive technologies (ART). Over the years, further research on the metabolism of pre-implantation embryos emphasized that there are specific needs depending on the developmental stage of the embryo (Johnson, 1988). This finding led to formulate the commercial sequential human embryo culturing media, such as G1 and G2 (Vitrolife), which we used in this study. Indeed, G1 supports the in vitro development of the fertilized oocyte up to the embryonic genome activation (EGA): 8-cell in human or rabbit embryos Kanka (2003), and G2 from EGA to blastocyst. 17

18 In contrast, single-step media, such as the Global (Life Global) medium we used in this study, are designed to provide all that the embryo needs from the fertilized oocyte to the blastocyst stage (Gardner and Lane, 2002; Gardner and Leese, 1990; Leese, 1988). Despite the efforts of the last century, some studies indicate that in vitro culture may adversely affect the developmental potential of the embryo or even have effects at later stages (postnatal or even adult) (Khosla et al., 2001). The in vitro culture technique is the most widely employed protocol in ART. However, compared to their in vivo counterparts, cultured embryos from all species have reduced pregnancy rates, reduced viability and growth, increased developmental abnormalities, behavioral deviations, metabolic disorders and exhibit aberrant expression patterns of imprinted genes (Schieve et. al.,2002; Khosla et al., 2001). A report from Greenblatte et al. (2005) showed that a more important percentage of human embryos cultured with Global medium reached the blastocyst stage compared to the ones cultured in G1/G2. However, there were no significant differences in terms of implantation and pregnancy rates between the two media after blastocyst stage. According to Market-Velker et al. (2010), one of the leading explanations for these culture-induced abnormalities are epigenetic alterations that originate from embryo manipulation and result in changes in gene expression. This study used three known marker genes for imprinting to compare five commercial media system. It demonstrated that maintenance of genomic imprinting is not better in sequential media compared to their singlestep counterparts. Therefore, our current study's aim was to investigate the dynamics of methylation and hydroxymethylation in rabbit embryos obtained from in vivo fertilization and developed in vitro in two different culture media (single-step: Global and sequential: G1+/G2+) in order to assess the potential effect of these media. Rabbit embryos were used as an advantageous model in the current study. It appears much more relevant than mouse embryos because its biological functioning is closer to human embryo. Indeed, a recent study performed in my host laboratory by Reis e Silva et al. (2012; 2011) revealed that in vitro culture has a significant effect on DNA methylation reprogramming during pre-implantation development in rabbit. 18

19 Moreover, the role of DNA methylation in genomic imprinting and the potential involvement of 5hMeC in gene activation as well as pluripotence maintenance make them particularly interesting to study. 19

20 2. Materials and Methods 2.1. Ethics statement The experiments were performed in accordance with the International Guidelines on Biomedical Research involving animals, as promulgated by the Society for the Study of Reproduction, and with the European Convention on Animal Experimentation. The researchers involved in work with the animals were all licensed for animal experimentation by the French veterinary services and the protocols were approved by the local ethics committee (Comethea) Experimental animals New Zealand White female rabbits (20-22 week-old) were used for embryo production in those experiments. All embryos were produced by natural fertilization. Superovulation was induced by means of 5 subcutaneous administrations of pfsh (Stimufol, Merial) for 3 days before mating: two doses of 5μg on Day 1 at 12-hour intervals, two doses of 10 μg on Day 2 at 12-hour intervals, and one dose of 5μg on Day 3, followed 12 hours later by an intravenous administration of 30 IU HCG (Chorulon, Intervet) at the time of mating (natural mating). The mating time served as the reference timings are subsequently noted as "hours post-coitum" (hpc) Embryo collection and culture Two different media were chosen for the experiments. Global medium is a single-step medium designed to provide all that the embryo needs from the 1-cell to the blastocyst stage. On the other hand, the sequential media G1+ and G2+ are designed to meet different needs between early and late cell stages, reflecting the environmental changes as the embryo journeys along the oviduct and into the uterus. Those two media are widely used in ART, especially in France and were thus of particular interest for this study. Prior to the experimental day, the Global (Life Global) and G1+ or G2+ (VitroLife) media were prepared for culturing and rinsing of embryos. Global medium was prepared with a ratio of 9:1 global medium and HSA (Human Serum Albumin, Life Global) respectively while G1+ and G2+ media were used directly as they already contain 10% HSA. 40µl drops of the different media were prepared under paraffin oil in a special culture dish (Falcon) and placed in an incubator at 20

21 37 C under 5% CO 2. In each experiment, embryos were recovered from three rabbits at 19hPC (1-cell stage). The oviducts were perfused with phosphate buffered saline (PBS, ph7.5, AMRESCO, Solon, OH). Immediately all embryos were transferred to 199 medium (Gibco) containing Hepes and kept at 37 C. They were then rinsed in the culturing media G1+ or Global. After all these environmental stabilizations were done, embryos were transferred into culturing media (G1+ and Global) prepared as stated above. Embryos were transferred from G1+ into G2+ at the 8-cell stage while embryos in Global medium were transferred into fresh drops of Global medium The experimental design for embryo development In each experiment, three to five cell stages were analyzed and at least two independent experiments were carried out for each cell stage and each of the analyzed marks. Additionally, to minimize the effects of genetic variations (the rabbits are genetically very close but not homozygous), embryos from three different rabbits were used each time and sorted equitably between the different media. In total, seven embryonic cell stages were used in the experiments: 2-cell (early and late), 4-cell, 8-cell, 16-cell, morula and blastocyst. In the case of the 2-cell stage, embryos were picked up every hour starting from the estimated onset of 2 cell stage (24hPC) and placed in a separate drop of medium. Then these embryos were fixed either after one hour for early 2-cell stage or after 4 hours for late 2-cell stage. The aim of separating those two groups is to determine whether the heterogeneity that exists at the 2-cell stage is due to the cell cycle. The timings corresponding to each developmental stage were determined prior to the immunostaining experiments, and the development of rabbit embryos in the used media until the blastocyst stage was assessed. The pickup and fixation time (in hpc) are presented in Table 1. Other groups: 4-cell, 8-cell, 16- cell, morula and blastocyst stages were fixed at 33, 46, 60, 72, 98 hpc, respectively. Blastocysts were hemi sectioned with a scalpel before fixation in order to increase the penetration of labeled molecules into the cells of the inner cell mass (ICM). Embryos were fixed with freshly prepared 4% paraformaldehyde (PFA) in PBS and kept at 4 C overnight. When the time between fixation and immunostaining was more than 48h, embryos were transferred from PFA to PBS and kept at 4 C until immunostaining could be started. 21

22 2. 5. Immunofluorescence All steps were performed in glass dishes and embryos were manipulated with glass mouthpipettes. Unless otherwise mentioned, all products were from Sigma-Aldrich, France and all steps were performed at room temperature. The embryos were washed in PBS for one hour with one change of PBS (2*30 minutes). Permeabilization was done with TritonX100, 0.5% in PBS, for one hour 15 minutes on a heating plate at 27 C. It was followed by saturation of non-specific sites with 2% bovine serum albumin (BSA) in PBS for one hour. Embryos were washed again in PBS-Tween % for 15 minutes followed by DNA denaturation with freshly prepared HCl 2N for one hour on a heating plate at 27 C. Embryos were ready for the primary antibody incubation after another rinse with PBS-Tween %. Then embryos were incubated at 4 C overnight in 20µl drops of primary antibody: anti-5-hydroxylmethylcytosine (5hMeC, rabbit polyclonal from Active Motif) or anti- 5-methylcytosine (5MeC, mouse monoclonal Eurogentec, BI-MECY 1000) with the dilution of 1:500 in PBS-BSA 2%. The antibody drops were prepared under paraffin oil in a petri dish. On the next day, embryos were washed with PBS-Tween % twice for 15 minutes. Then embryos were incubated for one hour in drops of secondary antibodies: Alexafluor488- conjugated antibody directed against rabbit (Jackson ImmunoResearch) for 5hMeC detection or fluorescein isothiocyanate (FITC)-conjugated antibody directed against mouse (Jackson ImmunoResearch) for the detection of 5MeC. Secondary antibodies were diluted at 1:200 in PBS-BSA 2%. From this step onwards, embryos were protected from light exposure. After one 30 minutes wash with PBS-Tween %, post-fixation was carried out with 2% PFA in PBS for 20 minutes. Finally, after a short rinse with PBS-Tween %, embryos were incubated with the DNA dye Ethidium Homodimer 2 (EthD-2) with the dilution of 1:500 in PBS-Tween 0.05% for 30 minutes in an incubator at 37 C. Embryos were then mounted on slides with Citifluor. 22

23 2.6. Protocol control To ensure that the signals detected for 5-hydroxymethylcytosine and 5-methylcytosine using our protocol were specific, antibodies against anti-5-hydroxymethylcytosine (5hMeC, rabbit polyclonal Active Motif 39769) or anti-5-methylcytosine (5MeC, mouse monoclonal Eurogentec, BI-MECY1000) were pre-incubated with either 1µM 2'-deoxy-5'-methylcytidine-5'- triphosphate (dm 5 CTP, Fermentas) or 1µM 2'-deoxy-5'-hydroxymethylcytidine-5'-triphosphate (Hydroxymethyl-dCTP, BIOLINE). Immunostaining experiments were then performed as described above using the pre-incubated anti-5-hydroxymethylcytosine or anti-5-methylcytosine antibodies. The optimal concentration for the primary antibodies was also determined by comparing signals obtained with different concentrations. Therefore, optimal concentration is in agreement with the range of Active motif recommendation (1:250-1:750) for anti-5hmec antibody dilution Imaging and analysis The embryos were observed and scanned for the area containing the nucleus using a Carl Zeiss AxioObserver Zl fluorescence microscope equipped with the ApoTome slider (MIMA2 Platform, INRA) equipped with an oil-immersion objective (Plan Apochromatic 63x, n.a.1.4). Digital optical sections were collected using a Z-series acquisition feature every 0.27 µm. Quantitative analyses of DNA methylation or hydroxymethylation level and total DNA contents were estimated by quantifying fluorescence signals using the Image-J software (National Institute of Health, Bethesda, Maryland, USA). To quantify the signal and the total DNA in the nuclei, we followed the next steps. 1) The area of each nucleus was outlined manually after a Z- projection of the acquired 3D-stacks summing the signal from each slice. 2) Then the mean signal fluorescence intensity and mean background intensity were measured for both 5hMeC/5MeC and DNA (EthD-2) images. 3) The corrected mean signal was calculated by substracting the mean background from the mean signal. 4) The corrected mean signal was multiplied by nuclear area to obtain the total corrected fluorescence intensity. 5) The corrected total intensity of 5hMeC/ 5MeC and EthD-2 were divided by the acquisition time of the corresponding signal. 6) Finally, DNA hydroxymethylation or methylation levels (corrected total fluorescence intensities per acquisition time for 5hMeC or 5MeC) were divided by the corresponding total DNA content (corrected total fluorescence intensities per acquisition time for 23

24 EthD-2) to obtain the normalized hydroxymethylated (5hMeC/EthD-2) or methylated (5MeC/EthD-2) DNA quantities Statistical analysis Experiments were replicated at least twice and data from each cell stage were normalized by the 4-cell stage median to reduce inter-replicate variability in staining intensities. Then all validated and corrected data from each experiment were merged to the corresponding cell stage under each culture medium for statistical analysis and graphical displays. Hydroxymethylated or methylated DNA per total DNA content was plotted using a box plot representation. The extreme outliers were omitted from the box plot. Because of our small set of data and to ensure its normality we used the log transformation version of our data for statistical analysis. SAS (2009) 9.1 version, ANOVA statistical procedure with the Tukey-Kramer multiple comparison was used to test whether there is significant difference of the DNA hydroxymethylated and methylated level among each cell stage. P-values < 0.05 were considered to be significant. 24

25 Figure 4: upper pannel, representative of the Z-projection images of 5hMeC immunostaining (green) and DNA labeling (EthD-2 red) at different developmental stages. Enlargement of nuclei (single slice) are shown on lower pannel (in black and white). Rings and patches of intense signal are indicated with arrows. White bar scale = 10µM. Table 2: Shows the numbers of nuclei used in the 5hMeC and 5MeC signal analysis for each cell stage in the two culture media are summarized as follows. Type of experiment Embryonic stages Number of analyzed nuclei Global G1+ /G2+ 2-cell Early cell Late cell hMeC 8-cell cell Total cell MeC 8-cell Total

26 3. Results I present here the data I obtained for 5hMeC and 5MeC immunostainings on rabbit embryos. The data on 5MeC is largely incomplete due to the difficulties we had to obtain the necessary biological material during my stay in the laboratory, which led us to cancel several planned experiments. We excluded one 5hMeC experiment, which contained morula and blastocyst stages due to the poor quality of immunostaining Dynamics of 5-hydroxymethylcytosine in rabbit embryos In this study, the dynamics of DNA hydroxymethylation was analyzed in rabbit embryos obtained from in vivo fertilization and developed in vitro. We used two different culture media (single-step: Global and sequential: G1+/G2+) as described in the material and methods section. The data I present was obtained from three independent experiments in which we collected a total of 384 embryos at 19hPC (1-cell stage) from nine rabbits, cultured them in the two culture media until the desired stage before fixation. The fixed embryos were subjected to an immunofluorescence protocol that allows preservation of the 3D structure. An anti-5hmec antibody was used to detect hydroxymethylation and DNA was counterstained with Ethidium Homodimer 2 (EthD-2). Embryos were then observed with an ApoTome (Zeiss) microscope. In all stages and in both media, we observed a diffused 5hMeC signal. At 2-cell late and 4-cell stages, we observed rings around the nucleoli precursors. At 8-cell stage patches were seen but not around the nucleoli. In addition, the signal was clustered to specific area in the nucleus. In general, the signal distribution was not uniform in the nucleus in both culture conditions in all stages. Examples of the images are shown on figure 4. We then quantified the 5hMeC signal. The number of nuclei analyzed for signal quantification at each cell stage in the two media is indicated in Table 2. To take into account the variations in the DNA content due to variations in the cell cycle, we also quantified the total DNA content and divided the 5hMeC signal by the DNA signal as explained in the material and methods section. In each experiment, we used the 4-cell stage as an internal control and divided all data by the median of the 4-cell stage. 26

27 Figure 5: Normalized DNA hydroxymethylation levels (5hMeC/EthD-2) in the nuclei of rabbit embryos developed in Global medium. n = number of nuclei analyzed at each stage. The asterisks (*) and arrows show significant difference (p <0.05) between two successive developmental stages. Figure 6: Normalized DNA hydroxymethylation levels (5hMeC/EthD-2) in the nuclei of rabbit embryos developed in G1+/G2+ medium. n = number of nuclei analyzed at each stage. Figure 7: Normalized DNA methylation levels (5MeC/EthD-2) in the nuclei of rabbit embryos developed in G1+ medium (left pannel) and Global medium (right pannel). n = number of nuclei analyzed at each stage. The asterisk (*) and arrow shows significant difference (p <0.05) between two successive developmental stages. 27

28 Normalized DNA hydroxymethylation levels in Global medium (Figure 5) significantly decreased between early 2-cell and 4-cell (p-value <10-3 ) and between 4-cell and 8-cell (p-value <10-3 ), but the observed difference between early and late 2-cell and late 2-cell and 4-cell was not significant. A significant increase of 5hMeC was observed between the 8-cell and 16-cell stages (p-value <10-4 ). In contrast, in the case of the G1+/G2+ culture media, the levels of DNA hydroxymethylation did not vary significantly (p>0.05) among the observed embryonic stages (Figure 6). Thus, the dynamics of 5hMeC in pre-implantation rabbit embryos appears different between the two culture media Dynamics of 5-methylcytosine in rabbit embryos We implemented similar protocol and culture conditions to analyze the dynamics of DNA methylation (5MeC) in rabbit embryo as described in the material and methods section. The data was obtained from two independent experiments in which we collected 117 embryos at 19hPC (1-cell stage) from five rabbits. An anti-5mec antibody was used to detect DNA methylation. The normalized 5MeC levels in the Global medium (Figure 7 right) did not vary significantly between 4-cell and 8-cells. A significant increase of 5MeC level (p-value <10-3 ) was observed between the 4-cell and 8-cell stages in the G1+ medium (Figure 7 left). Thus, as for 5hMeC, we observed different kinetics of 5MeC between the two media at the studied stages in rabbit embryos. 28

29 4. Discussion The present study provides the first quantitative analysis of 5-hydroxymethylcytosine (5hMeC) dynamics during the pre-implantation developmental period in rabbit embryos with comparison of two culture media conditions. The culture media used were Global and G1+/G2+ media, which are widely used for in vitro culture in ART worldwide and in particularly in France Methylation and hydroxymethylation dynamics up to the 8-cell stage In our analysis of DNA methylation and hydroxymethylation, we observed different kinetics between Global and G1+/G2+ culture media. Embryos developed in G1+/G2+ medium did not show any significant 5hMeC change across the considered embryonic stages, while we observed an increase of 5MeC between the 4-cell and 8-cell stages. In contrast, embryos cultured in Global medium showed a decrease of 5hMeC from the early 2-cell stage until the 8-cell stage while no significative change was observed for 5MeC between the 4-cell and 8-cell stages. Three hypotheses can explain the decrease of 5hMeC observed in Global medium. First, Inoue and Zhang (2011) showed that in mouse embryos 5hMeC can be lost by passive dilution, the level of 5hMeC is then decreased by half at each cell stage. Our data show that the ratio of 5hMeC per total DNA content (5hMeC/EthD-2) is reduced by half between the 2-cell, and the 4- cell stages ( ) and again between the 4-cell and the 8-cell stage ( ) in Global medium. Therefore, the decrease of 5hMeC between 2-cell and 8-cell stages could be explained by a dilution of 5hMeC during DNA replication at each cell cycle. Since, 5hMeC is reduced to half after each cell cycle; the newly synthesized DNA does not contain 5hMeC, which we could observe on mitotic chromosomes. Second, a recent study by Chen et al. (2012) has shown that the de novo DNMTs (DNMT3a and DNMT3b) have a DNA dehydroxymethylase activity in vitro and can directly convert 5hMeC to unmodified cytosine. However, as we did not observe an increase of 5MeC together with the decrease of 5hMeC in the Global medium, it is unlikely that DNMTs are involved. Whether the de novo DNA methyltransferase is involved in the decrease of 5hMeC with the increase of 5MeC, it could be tested by blocking all other possible pathways (TET, DNMT1, deamination, and DNA glycosylases BER) followed by the examining the kinetics of the two marks in vitro condition. Third, two recent studies from Ito et al. (2011) and He et al (2011) have shown that Ten eleven translocation (TET) proteins can oxidize 5hMeC 29

30 into 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Therefore, TET proteins could play a role in the decrease of 5hMeC we observe. It would be interesting to examine the dynamics of 5caC and 5fC between the 2 and 8-cell stage to see whether 5hMeC is oxidized in to 5caC and 5fC. In contrast with what is observed in Global medium, we did not observe any significant decrease of 5hMeC in the G1+/G2+ media. This could be due to a lower expression of the TET proteins. At the same time, we observed that 5MeC levels are stable in embryos cultured in Global medium and increase in those cultured in G1+/G2+ media. This suggests that DNMTs are active in those embryos, and are probably more active in G1+/G2+ media. This hypothesis could be tested by anti-dnmt immunostaining to determine the expression level of the DNMTs in both media. Those results are in contradiction with what had been observed previously in the host laboratory on embryos cultured in B2 medium or developed in vivo (Reis e Silva 2012). This may indicate that DNA methylation is particularly sensitive to in vitro culture conditions. However, we have only analyzed two stages and the experiments need to be further repeated to be able to draw firm conclusions DNA hydroxymethylation and Embryonic Genome Activation (EGA) Embryos developed in Global medium showed a significant increase of 5hMeC level between the 8-cell and 16-cell stages, and a small but insignificant increase was also observed in embryos cultured in G1+/G2+ media. The increase of 5hMeC could be associated with an over expression of TET protein. This hypothesis could be tested by hampering the expression level of the TET proteins followed by investigating the dynamics 5hMeC between 8-cell and 16-cell stages. Moreover, interestingly in rabbit embryos the genome major activation takes place between the 8-cell and 16-cell stage (Kanka, 2003). Several studies have shown a possible association between 5hMeC and transcriptional activation. For example, Ficz et al. (2011) have shown that 5hMeC is mostly associated with euchromatin and regions of active transcription, in contrast to 5MeC, which is linked to transcriptional repression. In addition, it has been suggested that the conversion of 5MeC to 5hMeC is a pathway to cancel the repressive effect of 5MeC on gene expression (Ficz et al., 2011). Finally, a study by Mellén et al. (2012) has shown that Methyl- CpG-binding protein 2 (MeCP2) is the major 5hMeC binding protein in the brain and that it makes the chromatin more accessible for gene expression. A similar mechanism could occur in 30

31 the embryo. Therefore, it would be interesting to test whether 5hMeC is involved in the genome major activation. To test this hypothesis, we could perform knock-downs (KD) of the TET proteins to block the conversion of 5MeC to 5hMeC, and use transcriptome analyses to determine the impact on the genome major activation Effect of in vitro culture In the present study we observed clearly an effect of culture condition on the dynamics of 5hMeC in rabbit pre-implantation embryos. The difference in kinetics of 5hMeC between Global and G1+/G2+ culture media could be associated with the nutrient composition of the culture media, which are vital for the embryo development. Thus, culture media components are linked with production of metabolite substrates, which have a connection with the donor of methyl group for the biological process of methylation (Zander-Fox and Lane, 2012). All DNA methyltransferases use S-adenosyl methionine (SAM) as the methyl donor (Bottiglieri, 2002). Actually we lack precise information on the composition of these commercial culture media to compare the ingredients at the formulation. However, as it has been stated in material and methods, Global medium is designed to provide all that the embryo needs from the 1-cell to the blastocyst stage. On the other hand, G1+ and G2+ are designed to meet different needs between early and late cell stages, reflecting the environment changes as the embryo moves along the oviduct and into the uterus. The rough composition of Global (LifeGlobal) medium is indicated in the annex. However, the composition for the G1+/G2+ (Vitrolife) medium is not available. Therefore, we cannot compare their nutritional component. Nevertheless, formulation of culture media may affect the metabolism of embryos and could be a reason for the observed difference between the two media. In conclusion, the role of 5hMeC is becoming an important topic in the study of epigenetic modifications, especially in early mammalian embryos. According to recent reports, 5hMeC could be involved in gene activation (Ficz et al., 2011) as well as maintenance of pluripotency (Tahiliani et al., 2009). Since we have shown that the dynamics of 5hMeC can be affected by in vitro condition, it would be interesting to assess its effect on gene expression. We need to clarify whether the rise of 5hMeC that we observed between the 8-cell and 16-cell stages is associated with the embryonic genome activation in rabbit embryos. We showed that the dynamics of 5hMeC is different in the two culture conditions Global and G1+/G2+ media. However, it is not 31