Epigenetic modification with trichostatin A does not correct specific errors of somatic cell nuclear transfer at the transcriptomic level; highlighting the non-random nature of oocyte-mediated reprogramming errors

Unless otherwise specified, all chemicals and media were obtained from Sigma-Aldrich
(St. Louis, MO, USA) and Gibco (Life Technologies, Rockville, MD, USA), respectively.
All animal care and surgical procedures were undertaken in strict accordance with
the approval of the Institutional Review Board and Institutional Ethical Committee
of Royan Institute (No#94321).

Experimental design

The objective of this study was to investigate the effects of TSA treatment of donor
cells on the transcriptional profile of cloned embryos compared to control SCNT and
IVF embryos (hereafter TSA-NT vs. CTR-NT vs. IVF, respectively) and to identify specific
genes or categories of genes that could account for specific mechanisms or events
that occur in somatic cells, reconstituted oocytes and developing embryos. To meet
these objectives, an established fetal fibroblast cell line obtained from an approximately
60-day female fetus, was treated with a previously established concentration and duration
of TSA (1.0 ?M for 24 h, 60]). At the end of each treatment, treated and untreated (control) cells were used for
either SCNT or flow-cytometery assisted analyses of DNA-methylation and histone acetylation.
Yield and quality of embryo development was recorded until day 7 when grade 1 2
blastocysts were used for microarray analysis, embryo transfer to recipients and differential
staining. Further, some of the SCNT and IVF embryos were collected at four intervals
post SCNT/IVF (0, 4, 8, 12, 24 h) for immunostaining assessment of epigenetic marks
and chromatin remodeling using immunostaining against 5-methylcytosine (5mC), 5-hydroxymethylcytosine
(5hmC) and ?-tubulin, respectively. Other embryos were also collected for the assessment
of nascent mRNA expression using immunostaining against BrUTP. Also, fibroblasts were
stably transfected with a construct of EGFP-OCT4 16] for the detection of the time-window of the expression of genes for pluripotency
in TSA-NT and CTR-NT embryos. Finally, qRT-PCR was used to determine the effects of
TSA on the expression profile of a subset of genes in fibroblast cells and resulting
embryos.

Embryo production by IVF

For in vitro maturation (IVM), cumulus-oocyte complexes (COCs) were obtained by aspirating antral
follicles (2–8 mm in diameter) of ovaries obtained from an abattoir. Groups of ten
COCs with homogenous cytoplasm and more than three layers of compact cumulus cells
were placed in 50-?l drops of maturation medium (MM) under mineral oil and matured
for 24 h at 39 °C under an atmosphere of 6 % CO
₂
in air with maximum humidity. The MM was comprised of tissue culture medium 199 (TCM199)
supplemented with 2.5 mM Na-pyruvate, 100 IU/ml penicillin, 100 ?g/ml streptomycin,
1 mg/ml estradiol-17?, 10 ?g/ml FSH, 10 ?g/ml LH, 100 ng/ml EGF, 100 ng/ml FGF, 0.1 mM
cysteamine, and 10 % fetal calf serum (FCS) at 39 °C, 6 % CO
₂
. 61].

For fertilization, commercially available frozen semen straws were thawed (30 s in
air and then 1 min in 37 °C water) and the semen was centrifuged (70 g for 5 min)
to remove the cryoprotectants. The semen pellet was layered upon a discontinuous PureSperm®
gradient (2 ml of 45 % over 2 ml of 90 % prepared in hepes TCM199 (HTCM199)) and motile
sperm were collected after centrifugation (1200 g, 20 min) at room temperature (RT).
Prepared samples were washed twice in hepes-buffered Tyrode’s albumin lactate pyruvate
medium and 2?×?10
⁵
sperm were co-incubated with ten COCs in 200 ?l fertilization medium for 18–20 h at
39 °C, 6 % CO
₂
and humidified atmosphere. The presumptive zygotes were then denuded of their cumulus
cells and washed twice with embryo culture medium. Since the SCNT procedure used in
this study was a zona-free procedure, in order to alleviate potential bias in the
array comparison of SCNT and IVF blastocysts, the zona pellucida was also removed
from IVF zygotes before culture for in vitro embryo development 16].

For embryo culture, a continuous modified synthetic oviduct fluid (mSOF) containing
100 ng/ml EGF, 0.5 mM glucose, and 2 ?l/ml ITS (insulin, transferrin and selenium)
was used. To preclude aggregation of zona-free embryos, 50-?l droplets of mSOF were
prepared under embryo-tested mineral oil in 3 cm Grainer® dishes. Next, ten small
wells were made by gently pressing a sterile steel rod with a round tip (500 ?m diameter)
to the bottom of the culture dish and the presumptive zygotes were put in separate
wells. The embryo culture dishes were incubated at 39.0 °C with 6 % CO
₂
, 5 % O
₂
, 90 % N
₂
and maximum humidity for 7 days. The numbers of embryos that cleaved and developed
to the blastocyst stage were recorded at day 3 and 7 post-IVF, respectively 16].

Embryo production by SCNT

For somatic cell culture, a skin biopsy from an approximately 60-day old female fetus
was used as the source of bovine fetal fibroblasts as described previously 60]. After thorough washing with phosphate buffer saline (PBS) containing antibiotics
and antimycotics, the biopsy was cut into small pieces (2–3 mm
²
), and the explants were cultured in Dulbecco’s modified Eagle’s medium F-12 (DMEM/F-12)
containing 10 % FCS and 1 % penicillin-streptomycin at 37 °C in a humidified atmosphere
of 5 % CO
₂
until confluence. To confirm the fibroblast phenotype, cells at passage two were immunostained
against anti-vimentin (for fibroblasts) and anti-pancytokeratin (for epithelial cells).
For TSA treatment, 4?×?10
⁵
fibroblasts at passage three were added to 3-cm culture dishes containing DMEM/F-12
plus 10 % FCS supplemented with either 1.0 (treatment) or 0.0 (control) ?M of TSA
and cultured for 24 h before being trypsinized for SCNT or flowcytometry assessment
of epigenetic marks as described elsewhere 60].

For oocyte reconstitution, denuded oocytes were released from their zona pellucida
by brief incubation (up to 45 s) in 5 mg/ml pronase dissolved in HTCM199 containing
10 % FCS. Enucleation of the oocytes was performed in PBS free of Ca
²⁺
and Mg
²⁺
and supplemented with 20 % FCS, 2.5 mM Na-pyruvate, 1 % bovine serum albumin (BSA),
1 % polyvinyl alcohol (PVA) and 1.5 mM glucose. Enucleation was carried out at 100X
magnification on a pre-warmed microscope stage (Olympus, IX71) under constant UV-light
exposure with the help of blunt perpendicular-break enucleation pipettes (15–20 ?m
inner diameter). Fibroblast cells were trypsinized immediately before NT, and a low
density of cells was prepared in a drop of HTCM199?+?0.5 % FCS containing 10 ?g/ml
phytohemagglutinin. Five to ten enucleated oocytes were added to the droplet, and
each oocyte was gently pushed over a single cell. The oocyte-donor cell couplets were
placed between two electrodes (0.5 mm apart), overlaid with a hypo-osmotic fusion
medium (0.2 M mannitol, 100 ?M MgSO
₄
, 50 ?M CaCl
₂
, 500 ?M hepes, and 0.05 % BSA), aligned first manually and then by application of
AC current (7 V/cm, 1,000 KHz, for 10 s). Fusion was induced by two successive DC
current pulses (1.75 KV/cm, 30 ?sec with 100 ?sec interval) 16]. Fused couplets were activated within 30 min of electro fusion by incubation in 5 ?M
calcium-ionophore for 5 min, followed by 4 h exposure to 2 mM 6-dimethylaminopurine
dissolved in TCM199 containing 10 % FCS, 0.2 mg/ml PVA, 3 mg/ml BSA 62]. Activated embryos were cultured and evaluated as described for the IVF embryos.

Quality assessment of blastocysts

For quality assessment of embryo development, blastocysts were graded according to
the guidelines of the International Embryo Transfer Society (IETS). Since it has not
been established whether these grading criteria are at all meaningful for zona-free
embryos or SCNT embryos, we also relied on our own experience 16], 18], 60]. For further assessment of embryo quality, differential staining to detect total
(TCN), inner (ICM) and trophectoderm (TE) cell numbers was carried out as described
previously 61]. In brief, blastocysts were incubated in 500 ?L of 1 % Triton X-100 and 100 ?g/mL
propidium iodide for up to 30 s depending on the size of the embryos, and then immediately
transferred to 500 ?l of a solution of 100 % ethanol plus 25 ?g/mL H33342 and stored
at 4 °C overnight. Fixed and stained embryos were subsequently mounted on glass microscope
slides in one drop of glycerol, gently flattened with a coverslip, and visualized
for cell counting on a fluorescence microscope (Olympus, BX51) using the 460 nm excitation
filter for blue and the 560 nm filter for red. TE cells were visualized as pink and
ICM as blue. The TCN of each embryo was calculated by adding the number of ICM and
TE.

RNA isolation, amplification, and microarray hybridization

The whole process of microarray analysis was as described previously 63]. Total RNA from each replicate was extracted and purified using the PicoPure RNA
Isolation Kit. After DNase digestion (Qiagen), the quality and concentration of the
extracted RNA was determined with a bioanalyzer (Agilent). All extracted samples were
of good quality with RNA integrity numbers ?7.0.

For microarray purposes, purified RNA was in vitro transcribed by T7 RNA amplification using the RiboAmp HS
^Plus
RNA Amplification Kit (Life Science) and labeled with Cy3 and Cy5 using the ULS Fluorescent
Labeling Kit (Kreatech). Antisense RNA (825 ng per replicate) was then hybridized
on the Agilent-manufactured EmbryoGENE® slides 30] in a two-color dye-swap design. The microarray chip used, EmbryoGENE®, covers most
of the bovine pre-attachment transcriptome and hence allows the analysis of gene expression
in bovine blastocysts.

After 17 h of hybridization at 65 °C, microarray slides were washed for 1 min in gene
expression wash buffer 1 (RT), 3 min in gene expression wash buffer 2 (42 °C), 10 s
in 100 % acetonitrile (RT) and 30 s in Stabilization and Drying Solution (Agilent).
Slides were scanned with a Power Scanner (Tecan) and features extraction was done
with Array-pro6.3 (Media Cybernetics). Intensity files were analyzed with FlexArray
1.6.1 (Michal Blazejczyk, Mathieu Miron, Robert Nadon (2007), FlexArray: statistical
data analysis software for gene expression microarrays. Genome Quebec, Montreal, Canada,
URL: http://genomequebec.mcgill.ca/FlexArray). Specifically, raw data were corrected by background subtraction, and then normalized
within and between each array (Loess and quantile, respectively). Statistical comparison
between treatments (CTR-NT vs. IVF and TSA-NT vs. IVF) was done with the Limma algorithm.
In pairwise comparisons among transcripts with a p-value??0.01, a false discovery rate (FDR) of 20 % and a fold change 2.0 were considered
differentially expressed. The data discussed in this publication have been deposited
in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession
number GSE57247 under URL: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE57247

Validation of microarray results by qRT-PCR

Microarray results were validated by quantitative real-time PCR (qRT-PCR) as described
previously 16]. The list of selected primers with their characteristics is shown in Additional file
9: Table S8. In brief, total RNA was extracted from independent samples (groups of
ten blastocysts in three replicates for each experimental group) using RNeasy Micro
Kit (Qiagen, Hilden, Germany) according to the manufacturer’s guidelines. The concentration
of the extracted RNA was determined by measuring the absorbance at 260 nm using a
spectrophotometer and total extracted RNA (around 0.5 ?g) was used for first strand
cDNA synthesis with the RevertAid® First Strand cDNA Synthesis Kit (Fermentas, Germany).
Each cDNA synthesis reaction contained 1 ?l random hexamer primer, 1 ?l RNase inhibitor,
4 ?l 5X reaction buffer, 2 ?l dNTP, and 1 ?l M-MuLV reverse transcriptase and was
adjusted to a total volume of 20 ?l using DEPC-treated water. The synthesis of cDNA
was performed at 42 °C for 1 h. The qRT-PCR was carried out using the Rotor Gene 6000
(Corbett®, Australia). Each reaction mix contained 2 ?l cDNA, 10 ?l SYBR Premix Ex
Taq II (TaKaRa®, Japan) and 1 ?l of 5 pM/ml forward and reverse primers and adjusted
to a total volume of 20 ?l using dH
₂
O. The primers were designed using Beacon Designer, checked by NCBI blast, and finally
by gel electrophoresis. The efficiencies of the designed primer pairs were checked
by amplification of a serial dilution of template cDNA (50, 10, 2, 0.4, and 0.08 ng)
and were between 90 % and 100 %. The obtained CT of each target gene was normalized
to the CT of a housekeeping gene (ACTB) and represented with reference to IVF as 2
^-??CT
.

Functional analysis of differential gene expression profiles

The Ingenuity Pathways Analysis (IPA; Ingenuity Systems, www.ingenuity.com) software was used to group overrepresented functions of differentially expressed
genes into clusters. Moreover, IPA was queried to compile canonical pathways as well
as gene regulatory networks (GRNs) that were differentially expressed between treatments.
We used IPA to build schematic representations of important pathways deregulated in
SCNT blastocysts 63].

Network generation

A data set containing the gene identifiers and the corresponding expression values
was uploaded into the application. Each identifier was mapped to its corresponding
object in Ingenuity’s Knowledge Base. A fold-change cut-off of 1.5 with a P value 0.05 was set to identify molecules whose expression was significantly differentially
regulated. These molecules, called network-eligible molecules, were overlaid onto
a global molecular network developed from information contained in Ingenuity’s Knowledge
Base. Networks of network-eligible molecules were then algorithmically generated based
on their connectivity. Molecules are represented as nodes, and the biological relationship
between two nodes is represented as an edge (line). All edges are supported by at
least one reference from the literature, from a textbook, or from canonical information
stored in the Ingenuity Pathways Knowledge Base. Human, mouse, and rat orthologs of
a gene are stored as separate objects in the Ingenuity Pathways Knowledge Base, but
are represented as a single node in the network. Nodes are displayed using various
shapes that represent the functional class of the gene product 63].

Canonical pathway analysis

Canonical pathway analysis identified the pathways from the IPA library of canonical
pathways that were most significant to the data set. The significance of the association
between the data set and the canonical pathway was measured in two ways: 1) a ratio
of the number of molecules from the data set that map to the pathway divided by the
total number of molecules that map to the canonical pathway was displayed; 2) Fisher
exact test was used to calculate a P value to determine the probability that the association between the genes in the
dataset and the canonical pathway would occur by chance alone. Green and red symbols
represented genes respectively down and upregulated in treated embryos compared to
controls. Gray symbols represented genes with significant expression in blastocysts
but with no difference between conditions, whereas white symbols represented genes
not present on microarray or with below-background intensity 63].

Immunofluorescence

DNA-methylation and histone H3K9 acetylation in fibroblasts

As described previously 60], quantitative assessment of DNA-methylation and histone H3K9 acetylation was conducted
by incubating fibroblasts with 1:400 and 1:200 dilutions of mouse anti-5-methylcytosine
and anti-H3K9 monoclonal antibodies, respectively. Fluorescein isothiocyanate-conjugated
goat anti-mouse immunoglobin was used as the secondary antibody at a 1:50 dilution.
A corresponding control for each experiment was included. Cells were filtered through
a 40-?m nylon mesh in order to exclude aggregated cells. Ten thousand cells were collected
with the fluorescence activated cell sorting (FACS)-Caliber and were analyzed using
CELL QUEST® 3.1 software (Becton Dickinson). Three replicates were conducted for each
treatment with appropriate controls to eliminate the possible effects of auto fluorescence
and non-specific binding by the secondary antibody.

DNA-methylation and histone H3K9 acetylation of blastocysts

As described previously 16], NT and IVF blastocysts were washed in PBS containing 1 mg/ml PVA and then fixed
in 4 % paraformaldehyde (PF) in PBS for 30 min. Permeabilization was carried out in
0.5 % Triton X-100 in PBS for 15 min. For DNA-methylation, embryos were first pre-treated
with 4 N HCl for 60 min at RT, and then washed with PBS. To preclude non-specific
binding of the primary antibody, embryos were treated with 3 % BSA in PBS for 60 min
at RT. These embryos were then incubated with the primary antibody: either mouse monoclonal
anti-5-methylcytosine (for DNA-methylation) (Eurogentec®, BI-MECY-0100) or mouse monoclonal
anti-H3K9 (for histone-acetylation), for 1 h at 37 °C, followed by three washes in
PBS containing PVA. Embryos were then incubated with the secondary antibody (goat
anti-mouse IgG-TRITC conjugate) for 60 min at 37 °C. Subsequently, embryos were re-fixed
overnight in 4 % PF and treated with 0.1 mg/ml RNase-A for 1 h before being washed
in PBS. The fixed and stained embryos were mounted and imaged as described for EGFP
fluorescence using 557 nm excitation and 576 nm emission filters. The median pixel
intensity of 10–15 nuclei was detected in each NT and IVF blastocyst. Briefly, five
different regions of each image that contained 10–15 nuclei were randomly selected
and the average pixel intensity of fluorescence emission in the nuclei located in
these regions was detected using Image J. software (National Institute of Mental Health,
Bethesda, Maryland, USA). The software was zeroed against background before use. Appropriate
controls were also included to eliminate the possible effects of auto fluorescence
of the first and second antibodies.

Chromatin remodeling dynamics

Stepwise assessment of chromatin remodeling was carried out as described previously
64]. In brief, reconstituted oocytes at 0.5–12 h post-reconstitution (hpr) were fixed
in 4 % PF for 15 min. Microtubules were immunostained with anti-?-tubulin monoclonal
primary antibody (1:100) and FITC labeled anti-mouse-IgG secondary antibody (1:100).
The chromosomes were counterstained with H333242 (2 mg/ml) and then samples were washed
and mounted on glass slides in glycerol droplets to be observed with an epifluorescence
microscope (Olympus, BX51) at 400X magnification. A digital image of each sample was
taken with a highly sensitive camera (Olympus DP-72) operated on DP2-BSW Software.

5mC and 5hmC dynamics

The disappearance of 5mC and appearance of 5hmC were assessed as described previously
65]. Briefly, reconstituted oocytes and IVF embryos at 0, 4, 8, 12, and 24 h post SCNT/IVF
were fixed in 4 % PF for 10 min at RT, washed several times in PBS-Tween (PBS-T: 0.1 %)
and then permeabilized in 1 % Triton X-100/PBS-T for 1 h. Tissues were washed again
in TBS-T then antigens were blocked by incubating in 5 % non-fat dry milk/TBS-T for
1 h at RT. First antibodies were added to fresh blocking solution at 1:1000 and 1:2000
dilutions for anti-5mC and anti-5hmC, respectively. Samples were incubated overnight
at 4 °C in antibody solution, and then washed in PBS-T several times. Goat anti-mouse
secondary antibody was diluted at 1:2000 in freshly made blocking solution. Samples
were incubated for 45 min followed by four washes of 15 min in PBS-T and incubated
in DAPI 1:1000 for nuclear staining. Samples were washed and mounted on glass slides
in glycerol droplets to be observed with an epifluorescence microscope (Olympus BX51)
as described for chromatin remodeling.

Nascent mRNA expression

Incorporation of BrUTP into embryos was performed as described by Bui et al. 26]. In brief, SCNT embryos at different stages of in vitro development (oocyte, 2-, 4-, 8–16 cells, compact morula and blastocyst) were washed
trice with electrical permeabilization buffer (PB: 0.25 M D-glucose, 100 ?M CaCl
₂
.2H
₂
O, 100 ?M MgSO
₄
, and 0.1 % PVP) and then incubated in transcription buffer (PB?+?10 mM BrUTP) for
1 min. Embryos were placed between two electrodes (0.5 mm apart) of an electro fusion
chamber filled with 20-?L droplets of transcription buffer and BrUTP internalization
was induced by two successive DC current pulses (250 V/cm, 80 ?sec with 1 min interval).
Embryos were transferred to mSOF medium two minutes after permeabilization and cultured
for 1 h. The embryos were then washed twice in PBS-PVA, fixed for 40 min in PBS-PVA,
washed twice in PBS-PVA and stored overnight at 4 °C in PBS supplemented with 3 %
BSA and 0.1 % Triton X-100. The embryos were then incubated for 2 h with mouse monoclonal
anti-BrdU (6 ?g/mL), washed twice in PBS-BSA before being incubated for 1 h in 1:200
dilution of Tritc-labeled goat anti-mouse IgG as second antibody. Embryos were then
washed out trice in PBS-BSA, 10 min each, and observed with high power inverted and
then epifluorescence microscopes as described above. Blastocysts and oocytes were
used as positive and negative controls, respectively.

Nascent EGFP-OCT4 expression

Fibroblasts were transfected with a previously produced EGFP-POU5F1 plasmid containing
a neomycin resistance gene via lipofection method (LipofectAMINE 2000
^TM
). The EGFP-POU5F1 vector map and the transfection protocol are available in a previous
article 18]. Stably transfected colonies, selected after antibiotic screening and PCR, were expanded
before being used for SCNT. The time window and the extent of EGFP-OCT4 expression
in the developed embryos at the 4- and 8–16-cell, morulae, and blastocyst stages were
assessed by inverted and then epifluorescent microscopy.

Identification of the effects of TSA on fibroblast gene expression

To identify the effects of TSA on fibroblast gene expression, microarray data were
queried to select a subset of non-DEG transcripts in both NT groups that were oppositely
expressed between CTR-NT and TSA-NT blastocysts. The primer list of selected genes
is shown in Additional file 9: Table S8. Then, based on a design described by Whitworth et al. 28], qRT-PCR was used to compare the expression profiles of this subset of genes in TSA-treated
and untreated fibroblasts, and CTR-NT, TSA-NT, and IVF blastocysts.