Claire Boulesteix

Nathalie Beaujean
email: nathalie.beaujean@jouy.inra.fr

Fluorescent immunodetection of epigenetic modifications on pre-implantation mouse embryos

Keywords: embryo, mouse, development, epigenetic, reprogramming, immunodetection, fluorescence, microscopy

published or not. The documents may come   L'archive ouverte pluridisciplinaire

1-Introduction

During normal development the two very specialized gametes, the sperm and the oocyte, undergo profound changes. They lose their characteristic nuclear and chromatin configuration giving way to the establishment of an embryo-specific chromatin and nuclear organization. It is exactly this distinctive embryonic configuration that should be achieved by the donor nucleus after nuclear transfer (NT) into an enucleated oocyte. This conversion of a differentiated nuclear organization into an embryonic one is forced by the intimate contact between the donor cell chromatin and the oocyte cytoplasm, allowing its "reprogramming". Although the mechanisms of this reprogramming remain poorly understood, it appears that the particular chromatin landscape established after nuclear transfer is crucial for embryonic genome expression and for further development ( 1234). Whereas the genetic information provides the framework for the manufacture of RNAs and proteins, chromatin structure and nuclear architecture indeed control the accessibility of proteins to the DNA, especially transcription factors and RNA polymerase, and thereby gene expression (5). It involves several mechanisms such as epigenetic modifications (DNA methylation or histone post-translational modifications for example) that can be examined by immunostaining (6). During the past decade, 3D immuno-detection on fixed NT embryo has therefore provided tremendous insight into the sub-nuclear positioning of proteins during these very first stages of development, and into mechanisms -such as the epigenetic ones modifying the degree of chromatin compaction -involved in the reprogramming of the donor cell (7). Fluorescent immunostaining allows the simultaneous detection and localization of two or three epigenetic modifications by combining primary antibodies produced in different species, and secondary antibodies with different fluorochromes. Moreover, immuno-detection makes it possible to have results even with a single embryo at the 1-cell stage, thereby reducing the problem encountered by the limited availability of embryos after nuclear transfer. The immunofluorescent-staining protocols usually used on thin cells in culture tend to flatten the specimens and are not optimal for the analysis of embryos. However by using alternative fixation procedures ensuring that the specimens maintain the 3D structure they had in vivo, 3D data sets can now be obtained on preimplantation embryos with a preserved shape (8,9). By combining this technique with high resolution microscopy -Structured Illumination Microscope or Confocal Laser Scanning Microscope-and precise image analysis it is also possible to quantify the epigenetic modification of interest and then to describe its evolution not only in term of presence or absence, but also in terms of localization and quantity during pre-implantation development (4,10-13).

Here, we describe the 3D fluorescent immuno-detection of epigenetic modifications in mouse embryos with 1) the fixation of the embryos, 2) the immunostaining itself and 3) the observation procedure.

2-Materials

For all the procedures some basic equipment is required (Fig. 1). 3), directly from the collection/culture medium to the fixative solution, with no rinse step (see Note 11). 5. Cover the glass dishes with the glass lids; in the case of a long fixation wrap each dish with parafilm to avoid drying out of the fixative at 4°C. 6. Incubate in PFA, either 20 minutes at room temperature (or 27°C on the heating plate) or overnight at +4°C.

3-2 Immunostaining

All steps are performed at room temperature (22-25°C or 27°C on the heating plate) with a total volume of 500µL per glass dish, unless otherwise mentioned. Between each step diligently follow hazardous waste recycling regulations. Carefully wash each glass dish with distilled water and dry it with a non-fluffy rag.

1. Transfer the embryos with the glass embryo manipulation pipettes from the fixative into PBS: a) if PFA incubation was ≤ 30 minutes, 2 times 5 minutes PBS rinse is enough b) if PFA incubation was longer, wash for at least 30 minutes (3 times 10 minutes). In both cases, and at each further step, embryos have to be aspirated in the pipette with a minimum amount of solution, breathed out in the next solution and moved within the glass dish several times (see Note 10) (Fig. 3). 2. For permeabilization, transfer the embryos and incubate 30 minutes in 0.5% TritonX100, freshly diluted in PBS (mix gently to avoid bubbles) (see Note 12).

Saturation of non-specific sites is performed by 1 hour incubation of the embryos in PBS

containing 2% BSA (PBS-BSA 2%) (see Note 13). 4. Dilute the primary antibody with PBS containing 2% BSA in an micro test tubes tube (see Note 14). You will need a total of 20µl per group + 20µl for equilibration. 5. In a Petri dish prepare 20µl drops of the primary antibody for each group and add one for equilibration (see Note 15). Cover the drops with mineral oil (Fig. 4). 6. Embryos are transferred group by group in the primary antibody: first in the equilibration drop and then in their respective antibody incubation drops.

3-3 Mounting the embryos on slides

1. Prepare SuperFrost Plus slides with identification of the experiment on the side (date/species/embryonic stage/number of embryos and antibody used) and as many areas as groups drawn on the back on the slide with a permanent pen (see Note 21 and Fig. 5 A). 2. Load a 100µl pipetman with 40µL of the antifading agent (see Fig. 5 B).

3-4 Microscope observation

A wide-field microscope is not adequate to capture thick specimens like three-dimensionally 3D-preserved embryos, as it does not have enough z-axis resolution. Thus 3D-preserved embryos should be observed either on a fluorescent microscope equipped with structured illumination or on a confocal laser scanning microscope. It is also necessary to use a high magnification objective adapted to fluorescent observations (X63 Plan-Neofluar for example). 1. Place the slide on the microscope with the cover slip on the same side as the objective. 2. Locate the embryos with a low magnification objective thanks to the drawings performed on the back of the slide. 3. Use high-magnification objectives to observe the immunostaining. Oil-immersion ones are usually better and require a small drop of immersion oil that should be place between the cover slip and the objective.

4. Choose the fluorescence wavelength and the emission/excitation filters corresponding to the antibodies used during the immunostaining procedure (see Note 25). 5. Adjust the parameters of the microscope (acquisition time for example) in order to obtain the best signal-to-noise ratio: saturation of the detector should be avoided and the background noise level should be kept to a minimum. 6. Perform Z-series acquisition with small z-steps in order to obtain the best 3D reconstruction afterwards (see Note 26). We also recommend taking 2-3 additional optical z-sections on top and below the limits of the staining (Fig. 6). 7. For comparison between groups and quantification purposes, the same objective, offset, amplifier gain, wavelength range, and dichromatic mirrors should be used for all embryos.

Notes

1. Ideally room temperature should be stable, between 22 to 25°C, otherwise we recommend using a heating block set at 27°C. 2. Aspirator tubes can be hand-made with HPLC clear tubing (diameter 2 millimeters) but are also commercially available (e.g. from Sigma-Aldrich). To this system you can add a filter tip and aspirate with mouth, or add a 1 or 2 ml syringe and control the valve with your hand. 3. To get embryo glass manipulation pipettes, stretch a glass capillary (or tip of a Pasteur pipette) in the flame in order to get very thin pipettes with diameters similar to the embryo ones. It is also easier to work with quite short pipettes (no more than 8 centimeters in total) and a little oblique. 4. Immunostaining is usually performed in commercially available glass dishes with one or several wells. Plastic 4-cell plates can also be used but are usually less convenient for small preimplantation embryos. The capacity of each well has to be ≥ 500µl. 5. Primary antibodies are recognizing specific antigens and can be isolated from various species (mouse, rabbit, rat, human). Some primary antibodies can already be chemically linked to a fluorophore (direct fluorescent antibody). This decreases the number of steps in the staining procedure making the process faster and can reduce background signal by avoiding some issues with antibody cross-reactivity or non-specificity. However, since the number of fluorescent molecules that can be bound to the primary antibody is limited, direct immunofluorescence is less sensitive. If the fluorescent signal is too weak, amplification of the signal may be necessary. Secondary components such as fluorescently-tagged secondary antibodies (indirect immuno-staining) can then be used: multiple secondary antibodies will bind a single primary antibody and provide a more pronounced signal. This also allows more flexibility because different secondary antibodies can be used for a given primary antibody. 6. In mouse preimplantation embryos we usually start with overnight incubation at 4°C but some antibodies may be incubated 1h at 37°C in an oven or a dry incubator. 7. An extensive assortment of nucleic acid stains is commercial available for DNA counterstaining of the embryos. Their fluorescence absorption and emission spectra span the visible-light spectrum from blue to near-infrared, making them compatible with many different types of secondary antibodies. We often use DAPI (blue), Yo-Pro-1 (green), Propidium Iodide (red) and Ethidium Homodimer 2 (red). 8. Glass cover slips are commercially available in a variety of widths, lengths, and thicknesses. We recommend square or rectangular ones, easier to place on the embryos, measuring 22*22mm up to 22*40mm. The thickness of the cover slip is crucially important for high-resolution microscopy. Most microscope objectives are designed for use with No. 1 cover slips (0.13 to 0.17 mm thickness). Use of cover slips that deviate from this thickness will result in spherical aberration and a reduction in resolution and image intensity. 9. A comparative experiment with a short fixation and a long one in parallel, using the same antibodies, should of course be performed to confirm that a long fixation step does not alter the immunostaining. 10. As embryos are known to be very sensitive to culture conditions and environmental changes, we advise to perform embryo fixation as quickly as possible and to transfer the directly from the collection/culture medium to the fixative solution, with no rinse step. 11. The global success of the immunostaining, especially the quality of the signal, resides in the perfect contact of the embryos with each incubation solution. It is thus necessary at each step to properly transfer the embryos and move them in the glass dish as described (Fig. 3). 12. In some cases, when the immunodetection is not giving a good signal, permeabilization may be either 1) extended to 1 hour, 2) performed with 1% Triton X-100, 3) performed at higher temperature, e.g. 37°C or 4) after removal of the zona pellucida by HCl or pronase treatments. This last step is very useful is other species than mouse. 13. If necessary you can add, after the saturation of non-specific sites, a step of denaturation with HCl (2N HCl at 37°C for 1 hour) in order to increase DNA accessibility to the primary antibodies. This is particularly important for antibodies directed against 5-MeC or 5-hMeC (14). 14. Antibodies have to be prepared in sterile conditions, either under an appropriate hood or next to a flame. The dilution of the primary antibody will be adjusted in preliminary experiments. The best is to determine the lowest concentration that still provides an immunostaining signal, with a good signal-to-noise ratio. You may also combine several primary antibodies if they are not derived from the same species (to avoid crossreactivity upon addition of the secondary antibodies) or if they are direct fluorescent antibodies. Combining several primary antibodies however requires preliminary experiments with each individual antibody to control that they do not physically crossreact with each other. In order for example to combine 5-MeC or 5-hMeC immunodetection with the detection of modified histones we recommend to proceed in several steps: 1) incubate with the primary antibody directed against the modified histone(s), 2) wash with 0.05% Tween-20 in PBS for 30 minutes, 3) post-fix in 4% PFA for 25 minutes, 4) treated with HCl as recommended in Note 13 and 5) incubate with the 5-MeC or 5-hMeC primary antibody. 15. If the immunostaining results are not satisfactory, showing too much background, especially big fluorescent dots due to antibodies aggregates, we recommend centrifuging the antibody (10 minutes at 13 000 rpm with a microcentrifuge) after the dilution, before taking the supernatant to prepare the drops. 16. Choice of the incubation length and temperature will be adjusted in preliminary experiments in order to find the conditions that provide the best signal-to-noise ratio. 17. 0.05% of Tween 20 may be added in these washing steps in order to reduce the background signal if necessary. 18. If you want to work on several proteins and use several antibodies simultaneously, make sure that 1) primary antibodies are produced in different species, 2) each corresponding secondary antibody is coupled to a different fluorophore and 3) that each chosen fluorophore can be distinguished by the microscope that will be used. Secondary antibody dilution will be selected according to the manufacturer's recommendations.

We commonly use 1/200 or 1/300 dilutions with antibodies from Jackson ImmunoResearch. 19. The "post-fixation" is used to preserve the immunostaining when immediate observation on the microscope is not possible. 20. DNA stains concentrations: 1µg/ml DAPI; 0.002 mM Ethidium Homodimer-2; 1µg/ml Propidium Iodide. 21. Do not use the extremity of the slide: when using an inverted microscope for observations the objective could be damaged. 22. Each step has to be done carefully in order to preserve the 3D-shape of the samples. 23. Be particularly careful to not dry the embryos. 24. Do not pushing on the coverslip in order to avoid squashing. 25. Fluorescence wavelength and emission/excitation filters can be found on the antibodies manufacturers' websites. We recommend the Fluorescence SpectraViewer from Invitrogen, Life Technologies that allows all kinds of combinations and comparisons. 26. The minimal z-step will depend on the resolution in the depth direction of your microscope that is determined by the numerical aperture of the objective, the refractive index of the objective immersion media and the wavelength of the light used. Mounting the embryos on slides has to be done carefully in order to preserve the 3D-shape of the samples. First prepare the following material: A) SuperFrost Plus slides with identification of the experiment on the side (date/species/embryonic stage/number of embryos and antibody used) and as many areas as groups drawn on the back on the slide with a permanent pen (do not use the extremity of the slide : when using an inverted microscope for observations the objective could be damaged); B) 100µl pipetman ready to use with 40µL of the antifading agent and two glass manipulation pipettes (a perfectly calibrated and a very thin one with a diameter smaller than the embryos); C) the immunostained embryos in their glass dishes, a glass cover slip, a micro-spatula and clear nail polish to secure the cover slip into place. Then follow the mounting steps: -

FIGURES

3 .

 3 Aspirate the DNA staining solution to halfway up the glass manipulation pipette and then aspirate 5 embryos of the first group, in a minimum volume of solution (see Note 22 and Fig.5 D step 1). 4. Breath out all the embryos, trying to put them in line in the delimited area of the slide (see Note 22 and Fig.5 D step 2). 5. Repeat the last two steps as many times as necessary, taking 5 embryos from the first group each time 6. In one hand, with a very thin glass pipette aspirate as much solution around the embryos as possible. In the other hand, be ready, with the pipetman containing the antifading agent to put down a little drop of antifading agent on embryos immediately after the removal of the DNA staining solution (see Note 23 and Fig.5 D step 3). 7. Repeat all the steps for the other groups of embryos. 8. Put a little drop of clear nail polish on each corner of the slide, next to the groups, in order to preserve the 3D-shape of the embryos (see Fig.5 D step 4). 9. Carefully put the cover slip on the slide helping yourself with a micro-spatula, starting from one side and getting down the other side slowly (see Fig.5 D step 5). 10. Secure with clear nail polish around the coverslip (see Note 24 and Fig.5 D step 6). 11. Keep the slides at 4°C until observation.

Figure 1 :

 1 Figure 1: Basic equipment required.Performing immuno-staining on whole-mount preimplantation embryos requires A) a dissecting microscope with appropriate working distance, B) an heating block set at 27°C and C) embryo glass dishes (with one or several wells) with glass lids.

Figure 2 :Figure 3 :

 23 Figure 2: Aspirator tube and glass manipulation pipettes. A) Examples of aspirator tubes with either syringe or mouth control and of some glass manipulation pipettes (straight and oblique) B) Choice of the right diameter for the manipulation of the embryos: it should fit exactly to the diameter of the embryos and have smooth extremity. If necessary the diameter can be increased by breaking carefully (with your nails or with a diamond pen) the extremity of the glass pipette.

Figure 4 :

 4 Figure 4: Antibody incubation.For primary and secondary antibody incubations prepare for each group (G1, G2, G3 etc…) 20µl drops of the antibody (diluted with PBS containing 2% BSA) and add one drop for equilibration (EQ). Cover the drops with mineral oil and transfer the embryos group by group: first in the equilibration drop and then in their respective antibody incubation drops.

Figure 5 :

 5 Figure 5: Mounting of the immunostained embryos on slides.Mounting the embryos on slides has to be done carefully in order to preserve the 3D-shape of the samples. First prepare the following material: A) SuperFrost Plus slides with identification of the experiment on the side (date/species/embryonic stage/number of embryos and antibody used) and as many areas as groups drawn on the back on the slide with a permanent pen (do not use the extremity of the slide : when using an inverted microscope for observations the objective could be damaged); B) 100µl pipetman ready to use with 40µL of the antifading agent and two glass manipulation pipettes (a perfectly calibrated and a very thin one with a diameter smaller than the embryos); C) the immunostained embryos in their glass dishes, a glass cover slip, a micro-spatula and clear nail polish to secure the cover slip into place. Then follow the mounting steps: -Step 1: aspirate the DNA staining solution to halfway up the glass manipulation pipette and then aspirate 5 embryos of the first group, in a minimum volume of the DNA staining solution. -Step 2: breathe out all the embryos, trying to put them in line in the delimited area on the slide. -Repeat steps 1 and 2 as many times as necessary, taking 5 embryos from the first group each time.-Step 3: in one hand, with a very thin pipette aspirate as much solution around the embryos as possible, however be particularly careful to not dry the embryos / in the other hand, be ready, with the pipetman containing the antifading agent to put down a little drop of antifading agent on embryos immediately after the removal of the DNA staining solution. -Repeat all the steps for the other groups of embryos.-Step 4: put a little drop of clear nail polish on each corner of the slide, next to the groups, in order to preserve the 3D-shape of the embryos. -Step 5: carefully put the cover slip on the slide helping yourself with a micro-spatula, starting from one side and getting down the other side slowly. -Step 6: secure with clear nail polish around the coverslip, without pushing on it in order to avoid squashing of the embryos.

  Figure 5: Mounting of the immunostained embryos on slides.Mounting the embryos on slides has to be done carefully in order to preserve the 3D-shape of the samples. First prepare the following material: A) SuperFrost Plus slides with identification of the experiment on the side (date/species/embryonic stage/number of embryos and antibody used) and as many areas as groups drawn on the back on the slide with a permanent pen (do not use the extremity of the slide : when using an inverted microscope for observations the objective could be damaged); B) 100µl pipetman ready to use with 40µL of the antifading agent and two glass manipulation pipettes (a perfectly calibrated and a very thin one with a diameter smaller than the embryos); C) the immunostained embryos in their glass dishes, a glass cover slip, a micro-spatula and clear nail polish to secure the cover slip into place. Then follow the mounting steps: -Step 1: aspirate the DNA staining solution to halfway up the glass manipulation pipette and then aspirate 5 embryos of the first group, in a minimum volume of the DNA staining solution. -Step 2: breathe out all the embryos, trying to put them in line in the delimited area on the slide. -Repeat steps 1 and 2 as many times as necessary, taking 5 embryos from the first group each time.-Step 3: in one hand, with a very thin pipette aspirate as much solution around the embryos as possible, however be particularly careful to not dry the embryos / in the other hand, be ready, with the pipetman containing the antifading agent to put down a little drop of antifading agent on embryos immediately after the removal of the DNA staining solution. -Repeat all the steps for the other groups of embryos.-Step 4: put a little drop of clear nail polish on each corner of the slide, next to the groups, in order to preserve the 3D-shape of the embryos. -Step 5: carefully put the cover slip on the slide helping yourself with a micro-spatula, starting from one side and getting down the other side slowly. -Step 6: secure with clear nail polish around the coverslip, without pushing on it in order to avoid squashing of the embryos.

Figure 6 :

 6 Figure 6 : Immunodetection of two epigenetic modifications in a 2-cell mouse embryo produced by nuclear transfer. Two different epigenetic modifications (H3K4me2/3 in red and H3K9me3 in green) are immuno-stained. DNA is counterstained in blue. The figure presents confocal sections according to the classical X/Y axis but also according to the X/Z axis showing the depth of the nuclei, at the position indicated by the white line. The 3D-shape of the nuclei is clearly preserved. The corresponding 3D reconstructions obtained with the 3D Viewer Plugin from ImageJ are shown on the right. It shows that whereas H3K4me2/3 staining is present in the whole nuclei, H3K9me3 aggregates preferentially on one side of each nucleus.

3-Methods 3-1 Embryo fixation

	1. Dissecting microscope with appropriate working distance
	2. Heating block set at 27°C (see Note 1) 1. Label the glass dishes and lids with appropriate signs in order to identify the different groups 3. Aspirator tube: about 40 centimeters length (see Note 2) (Fig. 2) (control, NT etc…), date and size (n=…). 4. Thin glass manipulation pipettes for embryos, usually home made in the flame with glass 2. Choose between the two types of fixation: capillaries or Pasteur pipettes (see Note 3) (Fig. 1) -a short one : 20 minutes at room temperature (22-25°C or 27°C on the heating plate) with 2% 5. Embryo glass dishes with glass lids (see Note 4) (Fig. 1) PFA freshly diluted in Phosphate Buffered Saline PBS, if you plan to perform
	immunostaining immediately after.
	2.1 Embryo fixation -a long one : overnight at +4°C with 4% PFA, freshly diluted in Phosphate Buffered Saline
	PBS, if you plan to perform immunostaining the day after. If necessary keep the embryos in
	1. pH indicator strips PFA for up to one week -in that case, change the PFA 4% every 2 days (see Note 9).
	2. Fixative: 20% paraformaldehyde (PFA) solution. Upon opening of the stock solution, 3. Prepare in each glass dish a volume of ~500µL PFA, diluted with PBS.
	adjust pH to ~7.0 with 0.2N NaOH if necessary and store maximum 1 month at 4 °C with 4. With a thin glass manipulation pipette transfer the embryos group by group in the
	light protection (aluminum foil for example). Before each experiment pH should be corresponding glass dish
	checked with a pH indicator strip: if lower than 7.0 or upper to 7.4, through it away.
	3. Phosphate Buffered Saline (PBS): prepare according to manufacturer's instructions,
	autoclave and store at 4°C. Filter through a 0.22-μm sterile cellulose acetate membrane
	before use.
	4. Parafilm
	2.2 Immunostaining
	Prepare all solutions using ultrapure water (18.2 MΩ•cm resistivity at 25 °C).
	1. Phosphate Buffered Saline (PBS): same preparation as above (step 3 in section 2.1)
	2. Permeabilization stock solution: 10% Triton X-100 stock solution is prepared with ultrapure
	water (w/v) and mixed slowly to avoid bubbles. Store at 4 °C for maximum 1 month.
	3. PBS-BSA 2% solution: dissolve embryo/culture tested BSA powder slowly in PBS (w/v)
	and filter through 0.22-μm sterile cellulose acetate membranes. Aliquots of 1mL or 2mL can
	be kept at -20°C for several months and thawed just before use. After thawing filter through
	a 0.22-μm sterile cellulose acetate membrane, store at 4 °C.
	4. Primary and secondary antibodies (see Note 5)
	5. Aluminum foil
	6. Petri dishes (35 or 60 mm diameter)
	7. 0.5 or 1.5mL micro test tubes
	8. Mineral oil, embryo tested: keep at room temperature and protect from direct light.
	9. 37°C oven or incubator (see Note 6)
	10. Nucleic acid stain (see Note 7)
	11. Mounting anti-fading medium
	12. SuperFrost Plus slides
	13. Glass cover slips (see Note 8)
	14. Clear nail polish
	15. Thin micro-spatula
	2.3 Microscope observation
	High resolution microscopy -such as Structured Illumination Microscope or Confocal Laser
	Scanning Microscope -is highly recommended.

(see Note 10) (Fig.

  . Second antibody incubation: dilute in PBS-BSA 2% (see Note 18), prepare 20µl drops under mineral oil in a Petri dish (see Note 15) and transfer the embryos group by group as above (first in the equilibration drop and then in their respective antibody incubation drops), incubate 1 hour at room temperature (22-25°C or 27°C on the heating plate). From this step, to the end, always carefully protect dishes from light, with aluminum foil for example.[START_REF] Beaujean | Effect of limited DNA methylation reprogramming in the normal sheep embryo on somatic cell nuclear transfer[END_REF]. Transfer the embryos in glass dishes of PBS. Wash 3 times 10 minutes to remove antibody excess (see Note 17). 11. Embryos can be transferred in 2% PFA (diluted in PBS) for 30 minutes + washed once with PBS (2 minutes) (see Note 19). 12. Transfer the embryos in glass dishes containing a nucleic acid stain diluted in PBS and incubate for 20 minutes at 37°C (see Note 20).

	9

[START_REF] Mason | Chromatin and epigenetic modifications during early mammalian[END_REF]

. Incubation in the first antibody can be done either 1 hour at 37°C or overnight at 4°C (see Note 16). If you are using fluorescent primary antibodies, directly go to step 10 after this incubation. 8. Transfer the embryos in glass dishes of PBS. Wash 3 times 10 minutes to remove antibody excess (see Note 17).

Acknowledgement

All the present and past members from the lab should be acknowledged for their hard work, especially Lydia Ruddick and Bénédicte Sanseau who corrected this book chapter. We are also grateful to Pierre Adenot and Renaud Fleurot for Confocal and Apotome microscopy on the MIMA2 platform (Microscopie et Imagerie des Microorganismes, Animaux et Aliments). Work in the lab is supported by the REVIVE Labex.


P Lessly

Sepulveda-Rincon

Edgar Del Llano Solanas

Elisa Serrano-Revuelta

Lydia Ruddick

Walid E Maalouf

Nathalie Beaujean
email: nathalie.beaujean@inserm.fr

Early epigenetic reprogramming in fertilized, cloned and parthenogenetic embryos

Keywords: Oocyte, Nuclear transfer, Embryonic genome activation, Histones posttranslational modifications, DNA methylation

Despite ongoing research in a number of species, the efficiency of embryo production by nuclear transfer remains low. Incomplete epigenetic reprogramming of the nucleus introduced in the recipient oocyte is one factor proposed to limit the success of this technique. Nonetheless, knowledge of reprogramming factors has increased -thanks to comparative studies on reprogramming of the paternal genome brought by sperm upon fertilization-and will be reviewed here. Another valuable model of reprogramming is the one obtained in the absence of sperm fertilization through artificial activation -the parthenote-and will also be introduced. Altogether the objective of this review is to have a better understanding on the mechanisms responsible for the resistance to reprogramming, not only because it could improve embryonic development but also as it could benefit therapeutic reprogramming research.

Introduction to Nuclear Reprogramming

The cells of an adult mammal show a striking variation in structure and function, conferred by the differential expression of tightly regulated and specific gene networks. With few exceptions, individual cell types have been shown to retain the entire genetic content of the totipotent embryo. Yet, specific gene expression patterns associated with differentiated cell states are highly stable and conserved after somatic cell division [START_REF] Margueron | Chromatin structure and the inheritance of epigenetic information[END_REF]. The process of restricting expression to lineage-appropriate subsets of genes is ongoing throughout development and is now understood to reflect an accumulation of "epigenetic" changes at specific gene loci [START_REF] Keenen | Chromatin Remodeling in Embryonic Stem Cells : Regulating the Balance Between Pluripotency and Differentiation[END_REF][START_REF] Roper | Defining pathways that enforce cell lineage specification in early development and stem cells[END_REF]. The term epigenetics, coined by Conrad Waddington in the 1940s, is now used to refer to "the study of changes in gene function that are mitotically and/or meiotically heritable and that do not entail changes in DNA sequence" [START_REF] Goldberg | Epigenetics: a landscape takes shape[END_REF][START_REF] Bonasio | Molecular signals of epigenetic states[END_REF]. These changes include the large scale positioning of chromosomes and genes within the nucleus as well as local modifications to DNA and chromatin [START_REF] Beaujean | Fundamental features of chromatin structure[END_REF][START_REF] Schneider | Dynamics and interplay of nuclear architecture, genome organization, and gene expression[END_REF]. Epigenetic changes affect the accessibility of DNA to the transcription machinery, hence, gene expression [START_REF] Beaujean | Fundamental features of chromatin structure[END_REF][START_REF] Schneider | Dynamics and interplay of nuclear architecture, genome organization, and gene expression[END_REF]. Local modifications include histone post-translational modifications (PTMs such as methylation, acetylation, phosphorylation, etc.), DNA methylation as well as remodeling of the chromatin [START_REF] Goldberg | Epigenetics: a landscape takes shape[END_REF][START_REF] Bernstein | The mammalian epigenome[END_REF]. Moreover, all these local modifications may specifically recruit factors, as in recruitment of bromodomain proteins to acetylated histones and of Chromobox (CBX) family proteins to methylated histones [START_REF] Kutateladze | SnapShot: Histone readers[END_REF].

Each differentiated cell type has a specific profile of epigenetic modifications at key loci, resulting in expression of only type-appropriate genes. Deviations from this profile in vivo are frequently associated with disease [START_REF] Portela | Epigenetic modifications and human disease[END_REF]. It is also increasingly recognized that deviations from normally observed epigenetic patterning can contribute to the altered cell behavior demonstrated by cancer cells [START_REF] Gal-Yam | Cancer epigenetics: modifications, screening, and therapy[END_REF]. On the other hand, alteration of these epigenetic modifications with the aim of conferring a more developmentally plastic cell state is referred as nuclear reprogramming, and is attempted experimentally via a number of different techniques [12,[START_REF] Halley-Stott | Nuclear reprogramming[END_REF]. The first amphibian and mammalian cloned animals were achieved by inserting a donor nucleus into an enucleated recipient oocyte [START_REF] Gurdon | Nuclear transfer to eggs and oocytes[END_REF]. In this approach (cloning by nuclear transfer or NT), the oocyte has to reprogram the injected nucleus, trying to mimic reprogramming of maternal and paternal DNA during natural fertilization (Figure 1) [START_REF] Loi | Asymmetric nuclear reprogramming in somatic cell nuclear transfer?[END_REF]. Mammalian nuclei have also been reprogrammed by transfer to the germinal vesicle of Xenopus oocytes [START_REF] Halley-Stott | Nuclear reprogramming[END_REF][START_REF] Alberio | Differential nuclear remodeling of mammalian somatic cells by Xenopus laevis oocyte and egg cytoplasm[END_REF] or by the fusion of donor cells with an "embryonic dominant" cell type [12]. These techniques employ the natural reprogramming abilities of oocytes, embryos and embryonic cells, without requiring knowledge of the precise factors required for reprogramming. However, as knowledge of reprogramming factors has increased, alternative techniques involving exposure of cells to specific combinations of transcription factors have grown in popularity. Nowadays, somatic cells can be virally transfected, at least in mouse, with no more than 4 key transcription factors (Oct4, Sox2, Klf4 and Myc) in order to induce pluripotency (Figure 1) [START_REF] Tanabe | Induction of pluripotency by defined factors[END_REF]. The availability of induced pluripotent stem cells (iPS cells) from different species is also increasing rapidly [START_REF] Ogorevc | Cellular reprogramming in farm animals: an overview of iPSC generation in the mammalian farm animal species[END_REF] although the underlying molecular mechanisms remain to be investigated. Specific combinations of transcription factors have also been used to switch directly from one cell type to another, a process known as transdifferentiation [START_REF] Ladewig | Leveling Waddington: the emergence of direct programming and the loss of cell fate hierarchies[END_REF].

The goals of this experimental nuclear reprogramming are twofold. Firstly, to elucidate the roles of different epigenetic marks (and associated protein complexes) in nuclear reorganization at fertilization and during development and, secondly, with the aim of developing applications that benefit to human health. Such applications include the reprogramming of readily accessible cell types such as dermal fibroblasts to produce cell lines (induced Pluripotent Stem cells or iPS cells) to be used for drug screening or study of disease pathways [START_REF] Ohnuki | Present and future challenges of induced pluripotent stem cells[END_REF][START_REF] Romano | A commentary on iPS cells: potential applications in autologous transplantation, study of illnesses and drug screening[END_REF]. These iPS cell lines could be used to select the most effective treatment for the individual patient, or for the production of cells and organs for autologous transplants without the ethical or immunological problems associated with allogeneic transplantation [START_REF] Ohnuki | Present and future challenges of induced pluripotent stem cells[END_REF][START_REF] Romano | A commentary on iPS cells: potential applications in autologous transplantation, study of illnesses and drug screening[END_REF].

As a research tool, nuclear reprogramming continues to yield insights into the mechanisms and complexes involved in differential control of gene expression [START_REF] Halley-Stott | Nuclear reprogramming[END_REF]. Despite this, and successful cloning experiments in a wide range of species, the efficiency of all techniques, as measured by proportion of nuclei leading to developmentally plastic cells or healthy adult animals, remains very low. Considering the possible therapeutic benefits of successful nuclear reprogramming, there is a great deal of interest in understanding the mechanisms responsible for this resistance to reprogramming.

Reprogramming at Fertilization

In mammalian species, the formation of the embryo begins with the fusion of two highly specialized haploid cells (sperm and oocyte) which gives place to a genetically new diploid organism: the zygote (or 1-cell stage embryo) with two haploid "pro"-nuclei, the paternal and the maternal one (Figure 1). The "early mammalian" or "pre-implantation" embryo development compresses the time from fertilization until the implantation of the embryo in the mother's uterus. During this period of development, epigenetic reprogramming of the genome inherited from the gametes is crucial [START_REF] Hales | Epigenetic Programming : From Gametes to Blastocyst[END_REF][START_REF] Beaujean | Epigenetics, embryo quality and developmental potential[END_REF]. Indeed, during the formation of gametes, both oocyte and sperm cells are subjected to epigenetic changes that permit the expression of specific genes required for germ cell development. As gamete maturation is near to completion, a reorganization of the genome occurs. Paternal genome becomes highly methylated and compact as histones are replaced by protamines [START_REF] Goudarzi | Genome-scale acetylation-dependent histone eviction during spermatogenesis[END_REF][START_REF] Boskovic | How mammals pack their sperm: a variant matter[END_REF]. On the other hand, the oocyte undergoes a chromatin restructuring from a non-surrounded nucleolus (open chromatin with few defined chromatin surrounding the nucleolus and transcriptionally active) to a surrounded nucleolus conformation (highly condensed chromatin with clear presence of chromatin around the nucleolus and transcriptionally silent) (Figure 2) [START_REF] Bouniol-Baly | Differential transcriptional activity associated with chromatin configuration in fully grown mouse germinal vesicle oocytes[END_REF][START_REF] Bonnet-Garnier | Genome organization and epigenetic marks in mouse germinal vesicle oocytes[END_REF] .

From fertilization, both the incoming paternal DNA complement and that of the oocyte itself are reprogrammed in a number of steps, resetting chromatin to the embryonic form capable of undergoing further changes required during development [START_REF] Andrey | Statistical analysis of 3D images detects regular spatial distributions of centromeres and chromocenters in animal and plant nuclei[END_REF][START_REF] Aguirre-Lavin | 3D-FISH analysis of embryonic nuclei in mouse highlights several abrupt changes of nuclear organization during preimplantation development[END_REF]. The defined epigenetic status of the previous gametes' genome must now turn into a whole new epigenome proper of an early embryo with totipotent capacity [START_REF] Beaujean | Epigenetics, embryo quality and developmental potential[END_REF][START_REF] Beaujean | Histone post-translational modifications in preimplantation mouse embryos and their role in nuclear architecture[END_REF][START_REF] Mason | Chromatin and epigenetic modifications during early mammalian development[END_REF]. To do so, the paternal and maternal genome undergo global demethylation and, although many studies have led to contrasted results regarding the dynamics and the extend of this demethylation [START_REF] Salvaing | Determinants of valid measurements of global changes in 5'-methylcytosine and 5'-hydroxymethylcytosine by immunolocalisation in the early embryo[END_REF], it appears that the demethylation process continues after the first cell cycle in the pre-implantation embryo up to the blastocyst stage in many mammals [START_REF] Salvaing | 5-Methylcytosine and 5-hydroxymethylcytosine spatiotemporal profiles in the mouse zygote[END_REF][START_REF] Reis | Dynamics of DNA methylation levels in maternal and paternal rabbit genomes after fertilization[END_REF][START_REF] Silva A E | Alteration of DNA demethylation dynamics by in vitro culture conditions in rabbit pre-implantation embryos[END_REF][START_REF] Yang | Epigenetic marks in cloned rhesus monkey embryos: comparison with counterparts produced in vitro[END_REF][START_REF] Beaujean | Nonconservation of mammalian preimplantation methylation dynamics[END_REF]. At this point, the first cell lineage determination takes place (the formation of the inner cell mass -ICM-and of the trophectoderm -TE) and new methylation patterns emerge together with cell differentiation and specialization until the whole organism is formed [START_REF] Silva A E | Alteration of DNA demethylation dynamics by in vitro culture conditions in rabbit pre-implantation embryos[END_REF][START_REF] Gao | Epigenetic regulation of gene expression in porcine epiblast, hypoblast, trophectoderm and epiblast-derived neural progenitor cells[END_REF]39].

In addition to this DNA demethylation occurring after fertilization, it has been shown in mouse that many of the histones replacing the protamines on the paternal genome are already acetylated like lysines 8 and 12 of histone H4 [START_REF] Worrad | Temporally restricted spatial localization of acetylated isoforms of histone H4 and RNA polymerase II in the 2-cell mouse embryo[END_REF]. Moreover, for a correct development, the paternal pronuclei has to be hyperacetylated with the further acetylation of lysines 5 and 16 of H4 and lysines 9, 14, 18 and 27 of histone H3 [START_REF] Mason | Chromatin and epigenetic modifications during early mammalian development[END_REF][START_REF] Adenot | Differential H4 acetylation of paternal and maternal chromatin precedes DNA replication and differential transcriptional activity in pronuclei of 1-cell mouse embryos[END_REF][START_REF] Santenard | Heterochromatin formation in the mouse embryo requires critical residues of the histone variant H3.3[END_REF]. On the other hand, some histone PTMs like trimethylation of lysine 20 on histone H4 (H4K20me3) and trimethylation of lysine 9 on histone H3 (H3K9me3, Figure 2), are inherited exclusively from the maternal pronucleus, creating an asymmetry between the two parental genomes in the embryo (it would not be possible to include in this work all known histone PTMs, their fluctuation and their roles; for a complete review of known histone PTMs see [START_REF] Beaujean | Histone post-translational modifications in preimplantation mouse embryos and their role in nuclear architecture[END_REF]). These asymmetries persist for varying lengths of time in the developing embryo. Per example, lysine 4 methylation on histone H3 is evenly distributed throughout DNA by the 2-cell stage [START_REF] Santenard | Heterochromatin formation in the mouse embryo requires critical residues of the histone variant H3.3[END_REF], while H3K9me3 remains asymmetrically distributed until the 4-cell stage [START_REF] Beaujean | Histone post-translational modifications in preimplantation mouse embryos and their role in nuclear architecture[END_REF]. Other modifications are found to differ from the ICM and TE cells, like H4/H2AS1P which is much frequent in the nucleosomes of TE than ICM cells [START_REF] Sarmento | Dynamic alterations of specific histone modifications during early murine development[END_REF], or the general methylation of H3K27 which is found only in the ICM, whereas in the TE it is only present in the inactivated X chromosome [START_REF] Erhardt | Consequences of the depletion of zygotic and embryonic enhancer of zeste 2 during preimplantation mouse development[END_REF].

The function of this asymmetry just after fertilization has not yet been fully understood, although it is thought to be required for a proper development. Indeed, embryos are transcriptionally silent until the end of the one cell stage, when a small number of embryonic genes are transcribed from the paternal genome [START_REF] Bouniol | Endogenous transcription occurs at the 1-cell stage in the mouse embryo[END_REF][START_REF] Aoki | Regulation of transcriptional activity during the first and second cell cycles in the preimplantation mouse embryo[END_REF]. This asymmetrical minor activation is followed by the major embryonic genome activation (EGA) later on, associated with a much more frequent rate of production of transcripts and the number of genes transcribed [START_REF] Aoki | Regulation of transcriptional activity during the first and second cell cycles in the preimplantation mouse embryo[END_REF][START_REF] Bensaude | Heat shock proteins, first major products of zygotic gene activity in mouse embryo[END_REF]. The reprogramming of histone modifications has been proposed to be significant for triggering transcription and EGA, correlating the accumulation of transcriptionally permissive marks on the paternal genome and minor activation, and between more widespread reprogramming and EGA [START_REF] Nothias | Regulation of gene expression at the beginning of mammalian development[END_REF][START_REF] Beaujean | Induction of early transcription in one-cell mouse embryos by microinjection of the nonhistone chromosomal protein HMG-I[END_REF][START_REF] Posfai | Polycomb function during oogenesis is required for mouse embryonic development[END_REF]. Among the differences observed in pre-implantation embryo between mammalian species, the timing of embryo genome activation is a major one. In mouse embryos, EGA occurs at two-cell stage, while in bovine and rabbit embryos it occurs at the eight-cell stage [START_REF] Duranthon | On the emerging role of rabbit as human disease model and the instrumental role of novel transgenic tools[END_REF][START_REF] Ménézo | Mouse and bovine models for human IVF[END_REF]. Remarkably though, it is believed that this the fourthfifth cell cycle in the bovine embryos is critical for chromatin remodeling and embryos that are unable to modify their chromatin structure for gene activation arrest at this stage. For example, distribution of H3K27me3 has been studied semi-quantitatively in bovine embryos, where levels were found to decrease from oocytes to their minimum at 8 cell stage, corresponding with EGA [START_REF] Ross | Polycomb gene expression and histone H3 lysine 27 trimethylation changes during bovine preimplantation development[END_REF][START_REF] Breton | Nuclear Profiles of H3 Histones Trimethylated on Lys27 in Bovine (Bos taurus) Embryos Obtained after In Vitro Fertilization or Somatic Cell Nuclear Transfer[END_REF]. The decline in H3K27me3 is independent of cell division, indicating an active removal mechanism, where histone demethylase KDM6B has been implicated as the enzyme catalyzing the removal [START_REF] Canovas | Jumonji domain-containing protein 3 regulates histone 3 lysine 27 methylation during bovine preimplantation development[END_REF]. Similarly, it appears that sheep oocytes and embryos have a specific Dnmt1 transcript involved in DNA methylation maintenance whose levels decrease when the embryonic genome becomes active at the 8/16-cell stage. Interestingly, reducing Dnmt1(12b) by RNA interference prevents embryo compaction at the morula stage, showing the importance of DNA methylation for embryonic preimplantation development [START_REF] Taylor | Cloning and expression of sheep DNA methyltransferase 1 and its development-specific isoform[END_REF].

Therefore, it seems that although the dynamics of some epigenetic marks are not conserved between all mammalian species, they are always closely related with the formation of an "open" chromatin state allowing gene expression regulation during preimplantation development

Reprogramming after cloning by Nuclear Transfer

Cloned embryos are the result of the enucleation of an oocyte and transfer of the diploid nucleus from another cell (Figure 1). After such nuclear transfer (NT) procedure, donor cell nuclei often get an incomplete reprogramming which is it thought to lead to abnormal development in clones [START_REF] Loi | Asymmetric nuclear reprogramming in somatic cell nuclear transfer?[END_REF]. In particular, the donor chromatin needs to undergo epigenetic changes and modifications in order to get an embryonic-like chromatin structure as seen in sheep, mouse, bovine and rabbit NT embryos [START_REF] Martin | Genome restructuring in mouse embryos during reprogramming and early development[END_REF][START_REF] Pichugin | Dynamics of constitutive heterochromatin: two contrasted kinetics of genome restructuring in early cloned bovine embryos[END_REF][START_REF] Yang | Heterochromatin reprogramming in rabbit embryos after fertilization, intra-, and inter-species SCNT correlates with preimplantation development[END_REF][60]. The timing and manner to achieve this conformation will depend on the type of cell used as donor for NT. Embryonic stem (ES) cells proliferate fast and appear to have a more open chromatin conformation than cumulus cells, which may have a more compacted genomic structure. This property seems to make the chromatin of embryonic stem cells more accessible to the cytoplasm of the recipient oocyte and to efficient reprogramming [START_REF] Maalouf | Trichostatin A treatment of cloned mouse embryos improves constitutive heterochromatin remodeling as well as developmental potential to term[END_REF]. Similarly, we observed that nuclear transfer of murine iPS cells results in higher rates of blastocysts and live-born cloned mice than embryonic fibroblasts (46% blastocysts and 1.3% liveborn for iPS cells versus 3.5% and 0% for fibroblasts, respectively) [START_REF] Liu | Induced pluripotent steminduced cells show better constitutive heterochromatin remodeling and developmental potential after nuclear transfer than their parental cells[END_REF]. Altogether, it seems that chromatin of the donor cells often remains too compact.

Trimethylation of lysine nine of histone H3 (H3K9me3) has been proposed to limit the success of nuclear reprogramming. H3K9me3 is indeed associated with the repression of transcription [START_REF] Hublitz | Mechanisms of transcriptional repression by histone lysine methylation[END_REF] and its localization has been shown to be strongly correlated with constitutive heterochromatin, where it recruits heterochromatin protein 1 (HP1β also called CBX1) [START_REF] Lachner | Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins[END_REF]. H3K9me3 distribution has also been revealed to significantly expand during the differentiation of human embryonic stem cells into fibroblasts, a process which involves spreading of heterochromatin [START_REF] Hawkins | Distinct epigenomic landscapes of pluripotent and lineage-committed human cells[END_REF]. Consistent with these observations, H3K9me3 has been shown to persist after bovine and mouse nuclear transfer experiments (Figure 2) [START_REF] Pichugin | Dynamics of constitutive heterochromatin: two contrasted kinetics of genome restructuring in early cloned bovine embryos[END_REF][START_REF] Zakhartchenko | Epigenetic marking correlates with developmental potential in cloned bovine preimplantation embryos[END_REF][START_REF] Ribeiro-Mason | Nuclear dynamics of histone H3 trimethylated on lysine 9 and/or phosphorylated on serine 10 in mouse cloned embryos as new markers of reprogramming?[END_REF] and H3K9me3 levels in lymphocytes have been correlated with decreased potential for nuclear reprogramming [START_REF] Baxter | Histone hypomethylation is an indicator of epigenetic plasticity in quiescent lymphocytes[END_REF].

A number of approaches have targeted H3K9me3 to improve nuclear reprogramming. In cell fusion experiments by Antony et al. [START_REF] Antony | Transient JMJD2B-mediated reduction of H3K9me3 levels improves reprogramming of embryonic stem cells into cloned embryos[END_REF], the transient induction of histone lysine demethylase KDM4D (also known as JMJD2B) in ES cells increased the proportion of cell reprogramming by 30% despite the rapid restoration of H3K9me3 levels thereafter. Similarly, the transient expression of KDM4D caused a twofold increase in the efficiency of reprogramming somatic cells into iPSCs [START_REF] Sridharan | Proteomic and genomic approaches reveal critical functions of H3K9 methylation and heterochromatin protein-1γ in reprogramming to pluripotency[END_REF]. Recently, it was shown that removal of H3K9me3 by overexpression of KDM4D can restore transcriptional reprogramming in mouse cloned embryos [START_REF] Matoba | Embryonic Development following Somatic Cell Nuclear Transfer Impeded by Persisting Histone Methylation[END_REF]. Such transient overexpression of KDM4D in cloned embryos has also been proven to efficiently improve reprogramming both in mouse and human cloning experiments, giving much higher rates of blastocysts [START_REF] Matoba | Embryonic Development following Somatic Cell Nuclear Transfer Impeded by Persisting Histone Methylation[END_REF][START_REF] Chung | Histone Demethylase Expression Enhances Human Somatic Cell Nuclear Transfer Efficiency and Promotes Derivation of Pluripotent Stem Cells[END_REF]. Histone acetylation is also very important for appropriate development in pre-implantation embryos. Studies regarding histone acetylation patterns in rabbit embryos [START_REF] Yang | Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation[END_REF] and bovine embryos [START_REF] Wu | Multiple histone site epigenetic modifications in nuclear transfer and in vitro fertilized bovine embryos[END_REF], produced either by in-vitro fertilization or somatic cell nuclear transfer, have shown significant differences. In vitro fertilized embryos always presented higher histone acetylation compared with their counterpart cloned embryos, underlying once again the compactness of chromatin after NT. The use of histone deacetylase inhibitors (HDACi), as Scriptaid or trichostatin A (TSA), to increase of acetylated histones and helping the chromatin opening in cloned embryos has been reported. The first successful group obtaining full-term developed embryos after NT from somatic cells was the group led by Dr. Kishigami [START_REF] Kishigami | Significant improvement of mouse cloning technique by treatment with trichostatin A after somatic nuclear transfer[END_REF], although at almost the same time another study was reported demonstrating that TSA could improve clone development [START_REF] Rybouchkin | Role of histone acetylation in reprogramming of somatic nuclei following nuclear transfer[END_REF]. An increase of the blastocyst yield and improvement of embryo quality after TSA treatment has been obtained with various donor cells: fibroblasts, neural stem cells, spleen cells and cumulus cells [START_REF] Inoue | Effects of donor cell type and genotype on the efficiency of mouse somatic cell cloning[END_REF]. It has also been demonstrated that this drug can help with gene expression regulation. For example, whereas cloned embryos showed a failure in the expression of Oct4an important factor for pluripotency maintenance-TSA treatment favored Oct4 expression in the correct number of cells at the blastocyst stage [START_REF] Eggan | Incomplete reactivation of Oct4-related genes in mouse embryos cloned from somatic nuclei[END_REF][START_REF] Hai | Pluripotency maintenance in mouse somatic cell nuclear transfer embryos and its improvement by treatment with the histone deacetylase inhibitor TSA[END_REF].

Thereafter, Scriptaid (SA) was reported to be a novel HDACi with less toxicity than TSA since it had a not lethal high efficiency even at high concentrations [START_REF] Ono | Inhibition of class IIb histone deacetylase significantly improves cloning efficiency in mice[END_REF]. Moreover, SA treatment could support full-term development of inbred cloned embryos. In fact, it appears that inhibition of HDAC is an important factor of reprogramming [START_REF] Kretsovali | Histone Deacetylase Inhibitors in Cell Pluripotency, Differentiation, and Reprogramming[END_REF]. Hence, the use of HDACi has resulted in significant improvements in cloning efficiency of many species including human [START_REF] Tachibana | Human embryonic stem cells derived by somatic cell nuclear transfer[END_REF].

Moreover, HDACi also favors global chromatin reprogramming and thereby gene expression in several species such as mouse or pig, by acting not only on acetylation of histones but also on H3K9me3 [START_REF] Bui | Effect of trichostatin A on chromatin remodeling, histone modifications, DNA replication, and transcriptional activity in cloned mouse embryos[END_REF] or even DNA methylation [START_REF] Hou | Effects of histone deacetylase inhibitor oxamflatin on in vitro porcine somatic cell nuclear transfer embryos[END_REF][START_REF] Xu | Effects of DNMT1 and HDAC inhibitors on gene-specific methylation reprogramming during porcine somatic cell nuclear transfer[END_REF]. HDACi improve genome-wide gene expression regulation bringing total gene expression profile of clones to resemble that of ferilized pups [START_REF] Kohda | Gene expression profile normalization in cloned mice by trichostatin A treatment[END_REF].We also demonstrated that addition of HDACi during the first cell cycle in cloned mouse embryos could improve nuclear remodeling of pericentromeric heterochromatin that reorganized around nucleolar precursors like in fertilized embryos [START_REF] Maalouf | Trichostatin A treatment of cloned mouse embryos improves constitutive heterochromatin remodeling as well as developmental potential to term[END_REF].Remarkably, the use of HDACi was also correlated with increased number of ICM cells and correct further development to term [START_REF] Maalouf | Trichostatin A treatment of cloned mouse embryos improves constitutive heterochromatin remodeling as well as developmental potential to term[END_REF].

Research on somatic cell nuclear transfer embryos has been very useful in portraying that these epigenetic modifications not only have the ability to alter the expression of genes, but also strongly demonstrate how their misregulation can disturb preimplantation embryonic development. Developmental inefficiency of cloned embryos and aberrant chromatin state seem to be tightly linked. The use of HDACi and of histone demethylases transient expression can however promote the formation of an "open" chromatin structure after NT, improving the reorganization of early embryo nucleus and thereby reprogramming.

Reprogramming in Parthenotes

Research in early mammalian development is carried out mostly on fertilized embryos. However, there is another way to study embryo development. Parthenogenetic activation is another valuable model to produce embryos in absence of sperm fertilization through the artificial activation of a metaphase II oocyte (Figure 1) [START_REF] Versieren | Developmental competence of parthenogenetic mouse and human embryos after chemical or electrical activation[END_REF]. In some species (like various fishes, ants, snakes or amphibians) parthenogenesis is a common method of asexual reproduction in which an unfertilized oocyte is able to develop into a whole new individual. Nonetheless, in mammals, parthenogenesis does not occur naturally, and if it does, it is only a consequence of erroneous oocyte maturation and embryos never develop to term [START_REF] Versieren | Developmental competence of parthenogenetic mouse and human embryos after chemical or electrical activation[END_REF]. In mouse, developmental arrest of parthenotes occurs before day 10 of gestation, but this time varies among species [START_REF] Brevini | Parthenogenesis as an approach to pluripotency: advantages and limitations involved[END_REF].

In normal conditions, ovulated oocytes advance from metaphase I to metaphase II and they remain arrested at this stage until they are fertilized by sperm. For the first cell division to occur, a series of events triggered by the entrance of a spermatozoon, known as oocyte activation, must take place. Broadly, the main trigger factor is the phospholipase-Ç (PLC zeta ) brought by the sperm into the oocyte's cytosol [START_REF] Ajduk | Cytoplasmic maturation of mammalian oocytes: development of a mechanism responsible for sperm-induced Ca2+ oscillations[END_REF]. A number of signaling pathways are then activated, which result in a calcium release inside the oocyte. This calcium increase is translated in the activation of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) which in turn, will inactivate the "cycle blocking" proteins maturation promoting factor (MPF) and cytostatic factor (CSF). The inhibition of these last two, releases the oocyte from its arrest and activation can be confirmed by the exocytosis of cortical granules, resumption of meiosis, extrusion of the second polar body and the formation of pronuclei. Without sperm, it is necessary to artificially induce oocyte activation if parthenogenetic embryos are to be obtained in the laboratory. There are different protocols capable of overcoming the arrested state of a metaphase II oocyte which may include temperature alterations, electrical pulses and changes in osmolarity [START_REF] Versieren | Developmental competence of parthenogenetic mouse and human embryos after chemical or electrical activation[END_REF]. Contemporary protocols are mostly based on calcium mobilizing compounds (i.e. ethanol, strontium or calcium ionophore) to foster the initial calcium release in the cytoplasm: protein kinase inhibitors or protein synthesis inhibitors (i.e. cycloheximide or 6-dimethylaminopurine) to inactivate the MPF and/or the CSF, and finally, a microfilament inhibitor (i.e. cytochalasin B) to avoid the extrusion of the second polar body [START_REF] Cibelli | Embryonic stem cells from parthenotes[END_REF][START_REF] Han | Effects of chemical combinations on the parthenogenetic activation of mouse oocytes[END_REF]. Indeed, avoiding the extrusion of the second polar body is necessary to maintain the diploidy in the future embryo (Figure 1) [START_REF] Bevacqua | Bovine parthenogenotes produced by inhibition of first or second polar bodies emission[END_REF]. Thus, diploid parthenotes only possess maternal genetic information and will be homozygous. In particular, diploid parthenotes will not present the two sets of maternal and paternal imprinted genes, reason why, mammalian parthenotes never develop completely unless genetically modified or by the production of chimeras with fertilized embryos [START_REF] Niwa | Nuclei of oocytes derived from mouse parthenogenetic embryos are competent to support development to term[END_REF][START_REF] Kono | Birth of parthenogenetic mice that can develop to adulthood[END_REF][START_REF] Jiang | Activation of paternally expressed imprinted genes in newly derived germline-competent mouse parthenogenetic embryonic stem cell lines[END_REF].

Therapeutically, since these embryos are not normally viable for full development, parthenotes are also being studied as a stem cell source as it would carry very few ethical issues [START_REF] Brevini | Parthenogenesis as an approach to pluripotency: advantages and limitations involved[END_REF]. Moreover, parthenotes are an effective tool to evaluate genetic effects on the process of maternal genomic imprinting [START_REF] Kono | Birth of parthenogenetic mice that can develop to adulthood[END_REF][START_REF] Latham | Genetic variation in trophectoderm function in parthenogenetic mouse embryos[END_REF]. They also offer a means to study the contribution of maternally derived factors, as well as the absence of paternal factors to early development. In NT experiments, oocyte activation is performed after nuclear transfer in order to induce the resumption of meiosis in the oocyte's cytoplasm. Comparing cloned embryos and parthenotes can therefore, be particularly helpful when it comes to study the precise cytoplasmic factors required for reprogramming within the recipient oocyte. Chromatin reorganization has been compared between fertilized embryos, clones and parthenotes in few studies. Parthenotes seem to have less problems than their counterpart cloned embryos in adopting the proper heterochromatin conformation at very early stages, at least in mouse and rabbit embryos (Figure 2) [START_REF] Yang | Heterochromatin reprogramming in rabbit embryos after fertilization, intra-, and inter-species SCNT correlates with preimplantation development[END_REF][START_REF] Merico | Epigenomic differentiation in mouse preimplantation nuclei of biparental, parthenote and cloned embryos[END_REF]. On the other hand, some epigenetic modifications take place more rapidly in parthenotes. Acetylation of histone H4 after formation of the pronuclei has been observed earlier in bovine and mouse parthenotes, probably due to the absence of the paternal genome [START_REF] Adenot | Differential H4 acetylation of paternal and maternal chromatin precedes DNA replication and differential transcriptional activity in pronuclei of 1-cell mouse embryos[END_REF][START_REF] Maalouf | Differential acetylation of histone H4 lysine during development of in vitro fertilized, cloned and parthenogenetically activated bovine embryos[END_REF]. Remarkably, we observed in a preliminary study that supplementation of TSA during the first embryonic cycle as in NT experiments resulted in an even more open chromatin structure in term of histone acetylation and in extended survival of mouse parthenotes post-implantation (unpublished data). All these observations make parthenotes an interesting model to study reprogramming by the oocyte's cytoplasmic factors, in the absence of any sperm supply.

Conclusion and Perspectives

Epigenetics is the area of molecular science which has been dusted off the shelves and gained a newfound interest. To facilitate a better comprehension of the complex interrelationships between all the various components of the epigenome, and the way that each individual part operates. It has been essential to decipher key elements of the nuclear reprogramming in early embryos. However, understanding the connection between chromatin structure, gene expression, genome organization, creation of the nuclear architecture, and how all of these cellular processes come together during embryogenesis still needs further studies. What it also needs to be remembered is that epigenetic changes can arise from external agents such as environmental cues, dietary, stress and chemical contaminants to mention some examples, which in turn, cause a chain effect to the chromatin modifying agents and their respective genes or gene families affecting normal development and disease through their actions on the epigenome [START_REF] Portela | Epigenetic modifications and human disease[END_REF]. This is particularly important from a clinical point of view. Indeed, the main goal in a fertility clinic is to raise embryos under the best culture conditions after gamete retrieving and in vitro fertilization to afterwards transfer the highest quality embryo to the mother's uterus and achieve a successful pregnancy [START_REF] Kupka | Assisted reproductive technology in Europe, 2010: results generated from European registers by ESHRE[END_REF]. This is nowadays an effective and common process thanks to all the research and advancements in assisted reproductive technologies (ART) which have been based on the knowledge obtained from studies mainly using mouse embryos because of their easy access and manipulation. Therefore, studies in early mammalian embryos (like mouse or rabbit) and their reprogramming could possibly help to improve embryo culture conditions to promote development of better quality embryos with higher potential for further development, thus increasing the success rates of ART [START_REF] Beaujean | Histone post-translational modifications in preimplantation mouse embryos and their role in nuclear architecture[END_REF][START_REF] Silva A E | Alteration of DNA demethylation dynamics by in vitro culture conditions in rabbit pre-implantation embryos[END_REF][START_REF] Duranthon | On the emerging role of rabbit as human disease model and the instrumental role of novel transgenic tools[END_REF]. Elucidation of the roles of epigenetic marks in nuclear reprogramming would also benefit human health, especially the reprogramming of iPS cells. In particular, some recent publications suggest that embryonic stem cells (ESCs) derived from cloned embryos may be closer to ESCs derived from in vitro derived embryos than iPS cells in terms of epigenome and transcriptome [START_REF] Tachibana | Human embryonic stem cells derived by somatic cell nuclear transfer[END_REF][START_REF] Ma | Abnormalities in human pluripotent cells due to reprogramming mechanisms[END_REF]. We hope better understanding of epigenetic remodeling mechanisms will shed some light on cell reprogramming and further application on stem cell therapies.

Figure 1 :

 1 Figure 1: Strategies used to induce nuclear reprogramming include (from left to right): induction by overexpression of embryonic pluripotent transcription factors, nuclear transfer of somatic cell nuclei into enucleated recipient oocytes, fertilization through sperm penetration and parthenogenesis by artificial activation.

Figure 2 :

 2 Figure 2: Examples of H3K9me3 immuno-staining (green) with DNA counterstaining (red) on nuclei from mouse oocytes in NSN (non-surrounded nucleolus) versus SN (surrounded nucleolus) oocytes and in 1-cell stage embryos: either fertilized (zygotes), cloned (obtained by nuclear transfer -NT) or parthenotes. Clear compaction of chromatin and accumulation of H3K9me3 can be observed in SN oocytes. After fertilization, asymmetric distribution can then be observed between the maternal and paternal pronuclei (mPN and pPN respectively) with H3K9me3 accumulation around the nucleolus precursor; whereas cloned embryos present no asymmetry with much more aggregates of H3K9me3, especially at the nuclear periphery. Bar= 10µm

Acknowledgements

Work in N. Beaujean's lab was funded by Laboratoire d'Excellence Revive (Investissement d'Avenir, ANR-10-LABX-73). PhD studies of LS Sepulveda-Rincon are sponsored by CONACyT Mexico and The University of Nottingham.