AABArchives Animal BreedingAABArch. Anim. Breed.2363-9822Copernicus GmbHGöttingen, Germany10.5194/aab-58-171-2015Quantitative analysis of RNA abondance for CTCF during reprogramming of bovine embryo from oocyte to blastocystAmiri RoudbarM.DehghaniH.TahmoorespurM.ZahmatkeshA.AdeldustH.Ansari MajdS.Daliri JoupariM.daliri@nigeb.ac.irDepartment of Animal Sciences, College of Agriculture, Ferdowsi University of Mashhad, Mashhad, IranDepartment of Basic Science, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, IranDepartment of Animal Sciences, College of Agriculture, Isfahan University of Technology, Isfahan, IranDepartment of Animal Science, College of Agriculture, University of Tehran, Tehran, IranDepartment of Animal and Marine Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, IranM. Daliri Joupari (daliri@nigeb.ac.ir)28April201558117117529January201426March2015This work is licensed under a Creative Commons Attribution 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/This article is available from https://aab.copernicus.org/articles/58/171/2015/aab-58-171-2015.htmlThe full text article is available as a PDF file from https://aab.copernicus.org/articles/58/171/2015/aab-58-171-2015.pdf
CTCF is a highly conserved protein among eukaryotes and it is involved in
many of regulatory functions including, transcriptional repression and
activation, chromatin insulation, imprinting, X chromosome inactivation,
higher-order chromatin organization, and alternative splicing. Studies
performed on mouse embryos indicate that CTCF can be a maternal-effect gene,
and is essential for normal development of embryos. CTCF can be used as a
molecular effector for the proper epigenetic establishment of embryonic
development. The aim of this study was to determine changes in transcript
levels of the CTCF gene in bovine preimplantation embryos. RNA was extracted from
immature and mature oocytes and embryos at various developmental stages (two-cell, four-cell, eight-cell, and blastocysts). Results showed that the amounts of
CTCF transcripts decreased in mature oocyte in comparison with immature
oocytes, but this change was not significant. In addition, the amount of
CTCF transcript in embryos at two-cell, four-cell, eight-cell, and blastocyst stages
significantly increased in comparison with immature oocytes. These data show
that CTCF expression in bovine embryo begins at minor embryonic genome
activation.
Introduction
Linear models of studies on gene expression has developed and changed into
three-dimensional models of gene expression profiling. These studies suggest the existence of factors that have roles in higher-order
chromatin organization. CTCF is a good candidate to play this important
role. These findings have led to recognizing this protein as a master
weaver of the genome (Phillips and Corces, 2009).
CCCTC binding factor (CTCF) is a highly conserved protein among eukaryotes,
which is expressed in almost all somatic cells similar to housekeeping
genes. CTCF is able to bind to a wide range of various sequences, and this
ability is based on combinatorial use of its different zinc finger domains
(Filippova et al., 1996). Existence of 39 609 binding sites in the genome of
mouse embryonic stem cells and 28 661, 19 308, and 19 572 binding sites in
the genome of human CD4+T cells, HeLa cells, and Jurkat cells,
respectively, for CTCF illustrates a very important role for this binding
protein (Cuddapah et al., 2009; Chen et al., 2008).
CTCF was discovered for the first time as a transcriptional repressor
(Klenova et al., 1993), and subsequent investigations
revealed that CTCF is able to act as a transcriptional activator factor
(Vostrov and Quitschke, 1997). Investigations of insulators,
which are important epigenetic regulators, showed that CTCF is necessary for
the performance of enhancer blocker (EB) insulators (Bell et al., 1999).
Methylation-sensitive CTCF binding sites are important segments for the
regulation of methylation at imprinted regions (Hark et al., 2000).
Interestingly, CTCF as a multifunctional factor regulates both
active and inactive X chromosomes (Chao et al., 2002; Pugacheva et al., 2005).
Alternative splicing plays a very important role during development and
differentiation. It has recently has been determined that there is a close
relationship between alternative splicing and epigenetic mechanisms
(Luco et al., 2011). Studies have shown that CTCF binds to the
unmethylated region of exon 5 of CD45 gene, and regulates alternative
splicing (Shukla et al., 2011). This very important finding provides a
new perspective of studies on this gene.
Despite the increase in our understandings of CTCF function in recent
years and the crucial role of this gene in epigenetic regulation, there is no
research describing CTCF expression pattern during the preimplantation period of
bovine embryos. Understanding CTCF expression patterns during
reprogramming, therefore, can help us improve our knowledge about gene
expression patterns during the preimplantation period of bovine embryos.
Accordingly, the aim of this study was to determine the amounts of CTCF
transcripts at various stages of embryo development using realtime PCR.
Material and methods
All reagents were purchased from Sigma Aldrich Company (Seelze, Germany)
unless otherwise specified.
Oocyte collection
Bovine ovaries and testes were collected from a commercial slaughterhouse.
These samples were transported to the laboratory in a standard saline
solution at 30 to 35 ∘C within 2 h. Cumulus-oocyte complexes (COCs) were
collected from 2 to 8 mm follicles by aspiration using an 18 gauge needle
and a
20 mL syringe. The COCs were selected according to morphological
characteristic (Wei et al., 2001) and washed three times in the
aspiration medium (TCM-199 supplemented with 3 mg mL-1 BSA, 20 mM sodium
pyruvate and 50 g mL-1 gentamicin sulfate). Some of the COCs were vortexed to
separate the cumulus cells from the oocytes. These immature oocytes were
washed three times with phosphate buffer saline (PBS) and were immediately
stored at -70 ∘C until the time of RNA extraction.
In vitro maturation (IVM)
Aspirated COCs were washed three times in the maturation medium (TCM-199
supplemented with 10 % fetal bovine serum (FBS), 20 mM sodium pyruvate, 50 ng mL-1 epidermal growth factor (EGF), 0.5 mg mL-1
follicle stimulating hormone (FSH), 5 mg mL-1 luteinizing hormone (LH), and 50 g mL-1 gentamicin sulfate). Groups of 10 oocytes were
incubated in droplets of 50 µL maturation medium under mineral oil for
23–24 h at 38.5 ∘C with 5 % CO2. After incubation, some of
the mature oocytes were vortexed to separate the cumulus cells. Oocytes were
washed three times with PBS and were immediately stored at -70 ∘C
until the time of RNA extraction.
In vitro fertilization (IVF)
Motile sperm cells were obtained from fresh epididymis of collected testes.
Sperm cells were washed two times in HEPES TALP medium and were separated by
the swim-up method. Some matured oocytes with expanded cumulus cells were washed
three times in TCM199 supplemented with 3 mg mL-1 BSA. Groups of 10 matured
oocytes were transferred into 50 µL droplets of fertilization medium
(IVF-TALP supplemented with 25 µg mL-1 heparin and 6 mg mL-1 fatty acid
free BSA). To each droplet, 10 µL of 2 × 106 sperm
concentration was added and plates were incubated for 22 h at 38.5 ∘C and 5 % CO2.
In vitro culture (IVC)
After fertilization, presumptive zygotes were denuded from cumulus cells by
slow pipetting. Zygotes were washed two times in the culture medium (human
embryo sequential media G1/G2 containing 10 ng mL-1 EGF and 5 % FBS). The first
group of 50 zygotes were cultured in four-well petri plates containing 250 µL
of G1 medium and incubated for 72 h at 38.5 ∘C and 5 %
CO2. After 72 h, embryos were transferred to another four-well petri plate
containing 250 µL of G2 medium and cultured for 4 to 6 days until
the
blastocyst stage. Samples of different embryonic stages including two-cell,
four-cell, eight-cell, and blastocysts were collected at various time intervals
(30–36, 36–72, 72–96 h, and 7–9 days respectively). Embryos were isolated
and washed three times with PBS and immediately stored at -70 ∘C
until the time of RNA extraction.
Relative abundance of CTCF in various stages of bovine
embryonic development. Data are shown as folds of transcript levels (2-log) ±SE. ns: not
significantly different from immature oocyte (P > 0.05), * and **:
significantly different from immature oocyte (P < 0.05 and P < 0.01,
respectively).
RNA extraction and RT reaction
Polled samples of 50 immature, 50 mature oocytes, 40 embryos at two-cell
stage, 30 embryos at four-cell stage, 20 embryos at eight-cell stage, and 10
embryos at blastocyst stage were used. All extractions of collected samples
(immature and mature oocytes, embryos at two-cell, four-cell, eight-cell, and
blastocyst stage) were done by Qiagen RNeasy® Plus Micro Kit
according to the manufacturer's procedure. DNase I treatment was performed
to remove genomic DNA from the samples according to the manufacturer's
procedure (Roche Applied Science). RNA was reverse transcribed to cDNA with
random primers and reverse transcriptase (Fermentas).
Realtime PCR and analysis of data
Realtime PCR was performed for CTCT as the target gene and H2A as an
internal control (all tests had three biological and technical replicates).
H2A was selected based on previous reports (Lonergan et al., 2003; Robert et al., 2002).
In this study two pairs of primers were used (Table 1).
Realtime PCR reaction was performed by Rotor-Gene 6000 (Corbett Research,
Australia), using Light Cycler® 480 SYBER Green I Master mix
kit (Roche Applied Science) according to the manufacturer's procedure. The
PCR procedure was performed by an initial denaturation at 95 ∘C
for 10 min followed by 40 cycles of denaturation for 5 s at 95 ∘C,
annealing for 10 s at 60 ∘C, and extension for 20 s at 72 ∘C for both genes.
In order to determine the amount of transcripts of the CTCF gene, raw data were
obtained from the system and CT information and amplification efficiencies
were determined using Linreg software (http://LinRegPCR.HFRC.nl) based on
baseline fluorescence collected during PCR (Ramakers et al., 2003).
Then these data were analyzed using REST software based on the Pfaffl method
(Pfaffl, 2001; Pfaffl et al., 2002).
Results
The amounts of CTCF transcripts at various stages of preimplantation embryos
were compared with immature oocyte (germinal vesicle GV) as control.
Comparisons are presented as expression ratio (2-log scale) ±SE
(based on biological replicates). In this study, the amounts of CTCF transcripts
in matured oocytes declined (not significantly) whereas embryos at
two-cell, four-cell, eight-cell, and blastocyst stage showed significant increases of
CTCF transcript levels (Fig. 1).
Discussion
Maternal-effect genes are necessary for normal embryonic development
(Wan et al., 2008). Depletion of CTCF by using transgenic RNAi mouse,
indicated that CTCF can be a maternal-effect gene. In mice, depletion of the
CTCF caused many of genes to become misregulated at early stages of
embryogenesis. These results indicated that CTCF has global and essential
roles in oogenesis and embryogenesis in mice (Wan et al., 2008; Moore et al., 2012).
Preimplantation development can be classified in different steps, including
first cleavage division, activation of the embryonic genome, compaction and
blastocyst formation. For the proper execution of these steps, the genes
should be expressed in a coordinated manner. Transcripts in oocytes or
embryos can be from the source of maternal or embryonic genome (Memili
and First, 2000). In mice, about 15 700 genes are expressed during
preimplantation development (Stanton et al., 2003), and the same number of
genes are expected to be expressed in cattle at this period. Precise
regulation of the expression patterns of these genes at the preimplantation
period requires correct reprogramming of the genome (Gondor and
Ohlsson, 2009). Our knowledge is quickly increasing about the expression
pattern of genes during preimplantation period. The proper expression pattern is
very important for the normality and quality of embryos
(Wrenzycki et al., 2005). Using a gene expression pattern is a
powerful tool to estimate embryo quality and viability at this sensitive
stage (Wrenzycki et al., 2007). According to recent
studies, CTCF was considered as a very important factor involved in many
parts of epigenetic regulation (Filippova, 2007) and, moreover, it
may act as a heritable factor for epigenetic regulation
(Phillips and Corces, 2009). Thus, CTCF can be used
as a molecular marker for the proper establishment of epigenetic mechanisms
during preimplantation development.
A very important event that occurs during mammalian embryonic development
is the replacement of maternal transcripts with embryonic transcripts. This is
performed in two phases of minor (after fertilization) and major (8- to 16-cell) embryonic genome activation (Badr et al., 2007). Data in
this study reveal that CTCF expression of the embryonic genome began at
minor embryonic genome activation, similar to mice at the two-cell embryo stage
(Wan et al., 2008). At major activation, the amount of the CTCF transcripts
were increased slightly. CTCF transcripts declined at maturation period,
probably due to the degradation of the maternal CTCF transcripts and
inactive embryonic genome.
CTCF has more than tens of thousands the binding sites throughout the mammalian
genome (Kim et al., 2007). The DNA-binding property of CTCF is sensitive to
methylation (Hark et al., 2000). In the reprogramming process, the whole
bovine genome undergoes gradual demethylation during the preimplantation
period until the 8- to 16-cell stages. After this stage, the genome undergoes de
novo methylation (Mann and Bartolomei, 2002). From
fertilization to 8- to 16-cell stages, we expect to witness that the binding
sites for CTCF to increase with demethylation. In our study, the amount of CTCF
transcripts increased after fertilization, which is probably coordinate with
the increase in binding sites for CTCF.
In this study, the amount of CTCF transcripts were obtained at in vitro
conditions. These changes may be the observed changes related to
environmental effects. The amount of transcripts in the early preimplantation
period can vary in different conditions, such as in vivo versus in vitro
(Lonergan et al., 2003). The quantitation of transcripts at in vivo conditions may be
a useful marker for showing expression differences in the two cases.
Acknowledgements
The authors would like to acknowledge the National Institute of Genetic Engineering
and Biotechnology (NIGEB, Tehran, Iran) for their financial support.
Edited by: K. Wimmers
Reviewed by: two anonymous referees