Acta Scientiae Veterinariae . 38(Supl 2): s509-s519, 2010.
ISSN 1678-0345 (Print)
ISSN 1679-9216 (Online)
Effect of nucleus transfer on gene expression in bovine embryos
during early development
Lleretny Rodriguez-Alvarez1 & Fidel Ovidio Castro1
ABSTRACT
Background: Somatic cell nuclear transfer, commonly known as cloning is a powerful tool for the production of
identical animals for productive, biomedical or conservation purposes. Today several species of mammals has been
cloned using this technology. Nevertheless, the efficiency of the process is still low in terms of newborn animals. The
causes for such inefficiencies are mainly the lack of knowledge about biological processes underlying this complex
technology particularly nuclear reprogramming to allow the establishment of embryonic gene expression pattern. The
abnormal phenotypes observed in clones during pregnancy and even after birth, have been associated with an
aberrant gene expression during early embryo development. A better understanding about the molecular process that
governs the early bovine embryo development could help to improve animal cloning efficiency. This paper reviewed
the effect of somatic cell nuclear transfer on gene expression in bovine embryos at both pre and peri-implantation
stages.
Review: This paper reviewed the effect of somatic cell nuclear transfer on early bovine embryo development. During
the early developmental period and prior to implantation, a bovine embryo passes through proliferation and differentiation
stages in order to assure implantation and foetation. Two stages have been described: pre-implantation stage when
the embryo reaches the blastocyst stage and peri-implantation also known as elongation stage. Those periods are the
result of a coordinated and robustly regulated gene expression patterns. In cloned bovine embryos as well as in other
species, several genes have been found deregulated not only during pre-implantation stages, but also during elongation.
Many of those genes such as OCT4, SOX2, NANOG and FGF4 are related with maintain of cell pluripotence, while
others such as INFtau, EOMES, CDX2 and TKDP1 are markers of trophoblastic function. Aberrant gene expression
pattern is not morphologically evident at blastocyst stage. However a deregulation of the expression of trophoblastic
markers during the peri-implantation period could be related with an incomplete morphological elongation. The pattern
of gene expression established as consequence of the reprogramming of the donor nucleus could change depending
on different factors inherent to the technique. Those factors include the donor cell, the cytoplast receptor and also the
technical process associated to nuclear transfer such as enucleation, cell fusion, and activation protocol. Despite the
multiples studies performed in order to improve cloned embryo competence in terms of gene expression pattern,
today this topic is still the milestone of many research groups.
Conclusion: Identification of deregulated genes may yield insights into mechanisms underlying the high mortality
rate of cloned bovine embryos, and lead to strategies to reduce embryonic losses.
Keywords: SNCT, gene expression, bovine, embryos, pre-implantation, elongation.
Running title: Gene expression in bovine embryos.
1
Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepción. Avenida Vicente Méndez 595, Chillán 537,
Chile. CORRESPONDENCE: [email protected]; [email protected] – TEL: +56-42-208835; FAX: +56-42-270212.
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I. INTRODUCTION
II. GENE EXPRESSION IN EARLY BOVINE EMBRYOS
III. GENE EXPRESSION IN PRE-IMPLANTATION BOVINE CLONED EMBRYOS
IV. GENE EXPRESSION IN PERI-IMPLATATION BOVINE CLONED EMBRYOS
V. CONCLUSIONS
I. INTRODUCTION
Somatic cell nuclear transfer (SCNT) in mammals is a complex process in which a differentiated somatic
cell is reprogrammed by soluble factors of an enucleated oocyte that will host said cell upon nucleus injection or
fusion. Since Dolly was born, animals from several species have been produced by SCNT turning cloning into an
attractive reproductive technology. In farm animals, SCNT first opened an opportunity to multiply genotypes of high
genetic value or animals from endangered species [1]. The option that offers this technology i.e. to modify the donor
cell before its transfer to an oocyte, represents an interesting way to produce transgenic animals for xeno-transplantation,
pharmaceutical protein production or as models to study different diseases [2,3]. After more than a decade of
experiments using a differentiated cell to produce a new individual, it has been proofed that SCNT is a powerful tool
to understand biological mechanisms as cell ageing, gene function, regulation of genomic imprinting and embryo
development, cell reprogramming as well as other topics [4].
Regardless the broad applications of animal cloning, its low efficiency producing healthy offspring is still
one of the most complex challenges of the technique. The efficiency of nuclear transfer could range between 1-10%,
what means that 1 to 10 live births will be obtained from 100 cloned embryos transferred to a surrogated mother [5].
In bovine, more than the 60% of the cloned fetuses are lost during the first trimester of pregnancy while less than 5%
of naturally produced fetuses are lost during this period [6]. Finally, a high rate of neonatal death has been observed
due to different pathologies [7].
After the nucleus transfer, the resulting cloned embryo should develop further under the command of the
former somatic cell to give rise to a genomic copy or clone of the individual from which the cell was extracted. During
this process, oocyte signals and factors shall erase and reprogram epigenetic markers of the donor nucleus and in
harmony with the nuclear DNA of the cell, reestablish appropriate gene expression patterns compatible with embryonic
development to term, including pre, peri and postimplantation development, placentation and birth. Gene expression
in cloned embryos is not always appropriately restored during reprogramming; altered expression patterns could be
responsible for the high incidence of pregnancy losses, the appearance of the so called Abnormal Offspring Syndrome
(AOS), as well as of other abnormalities in cloned fetuses and new born animals [8-11].
Therefore, the developmental potential of a clone derived from a somatic cell will be proportional to the
pattern of gene expression established as a consequence of nucleus reprogramming. Many strategies have been
used to improve the efficiency of cloning where most of them are focused on donor cell in order to make the nucleus
more suitable to be reprogrammed in an enucleated oocyte. Even though, the pattern of expression of developmentally
important and imprinted genes is very poorly understood and many experiments are yet to be conducted in order to
clarify which influence can have this process on gene expression and subsequently on the low success rate of animal
cloning. This paper aims to review the influence of somatic cell nucleus transfer on the gene expression pattern of
bovine embryos during the period prior to implantation.
II. GENE EXPRESSION IN EARLY BOVINE EMBRYOS
After fertilization and during the pre-implantation period, bovine embryos pass through proliferation becoming
expanded blastocysts around day 7 to 8. During this period, occurs the first decision about cell fate with the formation
of the inner cell mass (ICM) and the trophoblast (TB) [12,13]. The ICM mostly gives rise to the embryonic tissue
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whereas the TB gives extraembryonic tissues. At this stage the embryo has around 120 cells where 25% belong to
the ICM and 75% to the trophoblast [14].
Bovine blastocysts remain inside the zona pellucida up to day 9 after fertilization growing until approximately
200 µm and cell number can increase up to 160 [14]. After hatching, from the zona, embryos expand further and
change from spherical shape to a filamentous shape that reaches 150 mm by day 17 when maternal recognition of
pregnancy occurs [15-17]. Three steps of elongation are usually recognized in bovine: early or spherical (between day
9 and 12), ovoid (between day 12 and 14) and filamentous or late (from day 15 and prior to implantation [13]. The
trophoblast expansion provides an increased area to facilitate maternal-conceptus cross-talk necessary to allow
implantation [18]. This window of development is common in mammals with late implantation and it is known as periimplantation period. Implantation begins around Day 21 post fertilization [19], when the embryos reach a length of
around 300 mm [15-17,19-21].
These dramatic and rapidly occurring changes are tightly regulated at the gene expression level. At preimplantation stage, gene expression is characterized by two kinetic phases: 1) when the proteins are synthesized
from maternal mRNA accumulated during oocyte maturation and 2) when the embryo genome is activated and directs
the development. Of special relevance during this period is the expression of genes coding for pluripotence markers
as OCT4 (octamer-binding transcription factor), SOX2 (SRY (sex determining region Y)-box 2) and NANOG (Homeobox
transcription factor) that sustain the undifferentiated stage of the cells. In mouse blastocysts, OCT4 expression is
restricted to ICM and its expression is down regulated in trophoectoderm and special level of OCT4 are required to
maintain the pluripotence of the ICM cells [22,23].
In bovine species, OCT4 is expressed in both trophoblast and ICM and this pattern is maintained until the
filamentous stage [13,24,25]. Also OCT4 expression level did not change among blastocyst at Day-7 and Day-17
elongated bovine embryos [26]. The expression of NANOG and SOX2 in bovine embryos have been documented as
well; and their expression, similar to OCT4, was not silenced in the trophoblast cells of late elongated blastocysts,
suggesting a role of NANOG and SOX2 in the latest events of the peri-implantation development at least in cattle [13].
In cattle, differently to OCT4, the expression level of NANOG increases significantly from Day-7 to Day-17 [26].
Alike the genes mentioned above, FGF4 which transcript, in mouse blastocysts, encodes a trophoblast
growth factor is secreted only by the cells of the ICM. In bovine embryos it is also detected in the trophoblast at the
spherical stage but this expression is restricted to embryonic tissue at filamentous stages [13,27]. The expression
level of FGF4, decreases significantly from blastocyst to elongate stage at Day-17 of development [26]. This difference
in the expression pattern between mouse and bovine embryos is most likely in concordance with the active proliferation
of trophoblastic cells observed in bovine embryos during the elongation period [13]. In bovine embryos from blastocyst
stage (day-7) and during the onset of elongation, cells proliferate actively, while cellular interactions, cell-cell and cellmatrix signaling is predominant at the end of elongation [13,28].
It has been shown that interferon-tau (INFtau) [29], prostaglandin synthases (PGHS-2) [30], protease
inhibitors (TKDP family) [31] and pregnancy-associated proteins (PAG family) [32] are increasingly transcribed or
secreted when the bovine trophoblast elongates. During this period trophoblast cells differentiate, giving rise to the
mononucleated trophoblastic cells (MTC) and binucleated trophoblastic cells (BTC or giant cells). The MTC are the
major contribution of the trophoblast to the extraembryonic tissue and they are responsible for INFtau secretion which
is the first signal for pregnancy recognition in cattle. The BTC are only 20% of trophoblastic cells and they migrate
through fetus-maternal gaps to fuse with the uterine epithelium [33]. At late elongation and prior to implantation,
pregnancy-associated proteins and placental lactogen (CSHI) genes are expressed by the BTC [34]. The expression
of genes as INFtau, is evident as earlier as at blastocyst stage and the expression level of some of them changes
during elongation while in others remain constant [26,35,36]. This fact indicates that an accurate regulation of gene
expression occurs during early embryo development.
In bovine, little is known concerning the genes involved in trophoblast differentiation and most of the
research on this specie is based on the data from mouse embryos. In mouse blastocysts trophoblast specification
and maintenance are positively regulated through the action of transcription factors as CDX2 which specifies the
trophoblast versus the ICM cell fate and restricts the expression of OCT4 to the ICM cells [37]. Eomesodermin
(EOMES) controls the fate of polar versus mural trophoblast cells and ELF5 (member of an epithelium-specific
subclass of the Ets transcription factor family) regulate proliferation in the polar trophoblast lineage [37,38]. As in
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mouse, CDX2 have been reported to be expressed in bovine embryos at blastocyst stage [26,39] while ELF5 and
EOMES have been detected lately during peri-implantation stages [13,26].
Similar to this, MASH2 (mammalian achaete-scute homologue), a transcription factor which stimulates cell
proliferation and inhibits the differentiation of trophoblastic cells on giant cells (TGC), is expressed in the trophoblast
of bovine embryos at early stages but its expression level significantly increases at the end of elongation prior to
implantation [36,40]. Conversely, HAND1, also a transcription factor that in opposition to the function of MASH2
provokes differentiation of trophoblastic cells into TGC, is first detected at ovoid stage when trophoblastic differentiation
is evident [13].
The expression level of genes related with embryo metabolism such as the glucose transporters Glut-1
and Glut-3 as well as genes from the insulin-like growth factors system (IGF2, and receptors IGF-1r, -2r) also changes
during bovine embryo elongation [35]. In a transcriptomic experiment using a bovine-specific microarray, 26 genes
were down-regulated in bovine embryos between Day-7 blastocysts and Day-14 conceptus while more than 500
genes were up-regulated and most of them continued to be expressed until the time of implantation [28].
III. GENE EXPRESSION IN PRE-IMPLANTATION BOVINE CLONED EMBRYOS
After more than ten years of the first successful somatic cloning experiment and its potential applications,
the extensive use of somatic cell nucleus transfer is questionable due to the low efficiency reported in terms of
normal offspring that are obtained. From the very beginning, most of the efforts have been focused on improving the
efficiency of SCNT by increasing the percentage of good quality blastocyst that could be transfer to a surrogated
mother. In fact, at least in bovine, the actual level of blastocyst production by SCNT is similar or in some cases even
higher than the percentage of embryos obtained by in vitro fertilization [41,42]. Independently of this advance the final
efficiency is still the same or in the best case with a tiny increase.
The criteria for embryo selection before transfer are most of the time based on the embryo morphology
considering the presence of “nice” ICM and trophoblast, blastocyst expansion and cell number at blastocyst stage
[43]. The morphology classification for blastocysts selection is not an invasive method and it could predict the
pregnancy rate at least for in vivo produced embryos [44]. Despite the good morphology of the blastocysts produced
by SCNT only half of them are able to implant properly. In cloned embryos, it has been reported that many of the
genes that are crucial for normal development are aberrantly expressed both spatially and quantitatively in embryos
with an otherwise adequate morphology [23,45].
This fact indicates that the normal shape of the ICM and trophoblast is not sufficient to predict the capability
of the somatic nucleus to be reprogrammed and to establish the normal gene expression patterns supporting embryo
development to term. The abnormal gene expression in cloned embryos during pre, peri and post-implantation stages
are thought to be responsible for the low pregnancy rate and for the multiple disorders observed in the fetus and
offspring as well [8-11]. In this sense, the study of gene expression pattern at blastocyst stage becomes an interesting
approach to predict the reprogramming potential of a nucleus under specific conditions.
During SCNT the factors present in the receptor cytoplast will change the genome expression pattern of a
differentiated nucleus by the reprogramming process. The reprogramming of the differentiated nucleus should establish
the spatial, temporal and quantitative gene expression pattern in correlation with normal embryo development. The
molecular mechanisms underlying reprogramming are still unknown. DNA methylation, genetic imprinting and chromatin
remodeling are probably the processes, responsible for the establishment of gene expression pattern in cloned
embryos. In bovine clones many genes have been shown to be aberrantly express at blastocyst stage. These
changes in gene expression will go from no expression to up or down regulation. Most of those genes are related with
embryo processes such as compaction and cavitation, metabolism, stress response, early differentiation and
trophoblastic function and maintenance of pluripotence.
Theoretically the quality of a cloned embryo depends on a huge range of factors that for a better understanding
might be organized into two general groups: technical and biological factors. The technical factors are mainly referred
to the complicate steps of the technique, while biological factors refer to the quality and characteristics of the
recipient oocyte and of the donor cell. Despite this classification, these factors are strongly interconnected what
makes harder to elucidate the effect of each one on embryo development and specifically on nucleus reprogramming.
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From the technical point of view, SCNT is a complex technique that includes the enucleation of the recipient oocyte,
the fusion of the donor cell, reconstructed embryo activation and finally the in vitro culture of the embryo.
Many experiments on gene expression in cloned embryos have focused in finding the best combination of
expressed genes capable to assure an increase in the efficiency of cloning animals. From those experiments, clear
evidences of deregulation of gene expression pattern in cloned embryos have been obtained. However, there are also
contradictory results about the behavior of some specifics genes such as INFtau, OCT4 and imprinted genes as a
consequence of nuclear transfer [26,36,46-49]. This difference could be the effect of different experimental conditions
or little changes of the procedures.
For instance, Wrenzycki et al. [50] evaluated the effect of fusion and activation protocols on expression of
eight specifics genes in bovine blastocysts. Two groups of cloned embryos were included, in the first, cell fusion was
performed before activation (FBA) and in the second, fusion and activation were performed simultaneously (AFS). In
none of the groups, HSP70 (heat shock protein 70) mRNA was detected, in contrast with its expression in blastocyst
produce by in vitro fertilization (IVF). Conversely, INFtau was down regulated in FBA embryos compared with the
AFS and IVF counterparts. The expression of the other six genes (DcII (desmosomal glycoprotein desmocollin II), Ecad (E-cadherin), Glut-1, MASH2, IGF2r, DNMT (DNA methyltransferase)), was not different neither among the
cloned groups nor among clones and IVF embryos.
The expression of genes such as FGF4, FGFr2 and IL6 (interleukin 6), was not affected by the use of either
cell injection or cell fusion when the activation was performed at least 4 hours after reconstruction. The delay in the
activation after embryo reconstruction improved the efficiency of nucleus reprogramming by increasing the proportion
of embryos expressing FGF4 [51]. Even though, the number of bovine cloned embryos expressing FGF4 is significantly
lower than in the IVF counterpart and it correlates with their ability to establish pregnancy [51,52]. This could be
explained since in mice, FGF4, FGFr2 and IL6 play important roles at the time of implantation [53-55].
Until now most of the cloning experiments still rely on the original technique developed more than 30 years
ago when Willadsen et al. [56] produced sheep embryos by nuclear transplantation. The introduction of the “hand
made cloning” (HMC) to produce cloned animals have been a huge “jump” turning nuclear transfer in a easier and more
affordable technique since no expensive instruments and specials skills are needed [57]. Despite the benefits of
HMC there are some biological difficulties that should be faced. Probably the most significant task is that the mature
oocytes are released from the zona pelucida to facilitate the enucleation what is done by hand using a microblade
instead a pipette and micromanipulators.
During pre-implantation stage, embryos develop inside the zona pelucida conferring mechanical protection
and maintaining the tight junction between blastomeres. All this suggest that this structure is critical for normal
development, however, zona-free bovine and pig embryos produced by HMC have resulted in the birth of healthy
offspring [52,58-62]. Using HMC, the rate of embryo development to blastocyst stage is similar and even higher
compared with the traditional SCNT as well as is the pregnancy rate [52,63]. Little is known regarding the influence of
HMC on gene expression in pre-implantation embryos and no data comparing gene expression in embryos produce
by HMC and traditional SCNT using the same conditions (cell line, oocyte source, fusion and activation protocols and
embryo culture) are available. Nevertheless, primary data suggested that HMC will affect expression pattern of genes
related with both keeping pluripotency and trophoblast function in bovine cloned embryos comparing with in vitro and
in vivo produced embryos [26,39,52].
From the biological point, the donor cell has been the major focus of the studies concerning improvement
of SCNT. In this sense, different aspects have been studied including: cell type [64,65], tissue origins [66-69], animal
donor age [42,70-72], cell cycle stage and different treatment to induce it [64,73-79], synchrony between donor cells
and recipient oocytes [77,80], number of passages [81], cell culture and cell store conditions [82,83]. More recently,
the use of epigenetic modifiers becomes an interesting approach to make donor nucleus more suitable to be
reprogrammed [84,85].
Independently of the extensive experiments reported about the conditions of donor cell there is still not a
consensus about which combination is the best to improved nucleus reprogramming, even more, the results some
times are contradictory. For instance, fetal cell lines have been chosen as the best candidate over adult cells to
improve embryo development since its biological age is closer to an embryo and consequently, nucleus reprogramming
will be easier [1]. Furthermore, a higher incidence of pregnancy lost has been observed when adult cell lines are used
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instead fetal cell lines as nucleus donor [71,72]. Differently, in another case, adult cells produced a higher number of
blastocysts and seem to be more susceptible to nuclear reprogramming compared with a fetal line [42].
Bovine blastocysts produced by HMC using an adult cell line have shown to have a more similar gene
expression pattern to IVF embryos than cloned embryos produced using a fetal cell line as a donor nucleus. In fact
the number of embryos expressing NANOG, SOX2, and FGF4 was higher in adult cell-derived embryos and significantly
differences in the expression level of INFtau and OCT4 compared with IVF embryos was neither observed in those
embryos [26]. This result is in agreement with the high potential of those adult cell-derived cloned embryos to develop
until blastocyst stage compared with the fetal cell-derived counterparts (65 and 38 % respectively) [42]. In this
experiment OCT4 expression was significantly higher in cloned embryos produced with the adult cell line compared
with both fetal cells nuclear transfer-derived embryos and IVF embryos. Interestingly, OCT4 expression was detected
in the adult cell line use as donor nucleus. It seems likely that regulatory mechanisms of the growing blastocysts are
unable to compensate the constitutive levels of OCT4 present in the cells. No over expression of OCT4 has been
reported in cloned embryos using somatic cell as nucleus donor, but taken in account the high development potential
of those embryos the OCT4 expression level might be not detrimental [26]. Similar to these results Bortvin et al. [86]
showed that mouse blastocysts produced by nucleus transfer from ES cells which expressed OCT4 were able to
rescue OCT4 expression in 100% of the cases, while somatic cell clones from cumulus cells, could only partially
reprogram OCT4 expression.
Daniels et al. [51,87] showed as well that the expression of a specific gene in the donor cell will facilitate the
expression of that gene in the resulting embryo after nuclear transfer and viceversa. These results could explain why
certain cell types are more suitable to be reprogramming independently of their origin. Many researches support this
hypothesis since different cells lines used as a donor nucleus will produce cloned embryos with different gene
expression patterns that also reflect the development potential of those embryos [26,51,88,89]. All this also suggest
that each cell line even originated from the same animal could have different potential to be reprogrammed depending
on their epigenetic status. Identifying specific markers in the cell line and their correlation with high embryo development
potential would provide and interesting tool to improve nuclear transfer efficiency.
IV. GENE EXPRESSION IN PERI-IMPLANTATION BOVINE CLONED EMBRYOS
Despite the big amount of information gathered from gene expression profiles of pre-implantation embryos,
little is still known about the influence of differential gene expression of developmentally important genes in regard to
implantation and embryo-maternal interactions during the elongation period in bovine embryos. Especially, scarce is
the literature regarding this critical period of early development after SCNT. This is probably due to the difficult and
expensive procedures used to obtain elongated embryos in vivo and the lack of alternative in vitro techniques. As it
was previously mentioned in this review, in bovine the peri-implantation period is a critical phase where many changes
occur in both embryo and endometrium to allow implantation. During this time the embryo releases many factors that
act as the primary signal for maternal recognition. The expression of many of those factors starts during elongation
while the expression level of others changes during this period.
Using a semi quantitative PCR approach several trophoblastic markers have been found deregulated in
elongated bovine cloned embryos (Day-17) while others are normally expressed. Arnold et al. [36] detected an up
regulation of MASH2 in fetal fibroblast cells-derived bovine cloned embryos while the expression of PAG-9 and
HAND1 was down regulated in those embryos compared with both in vivo and in vitro counterparts. In the same
experiment the expression of INFtau was significantly higher in cloned embryos. On the contrary, INFtau was higher
in bovine cloned embryos produced using adult somatic cell as a nucleus donor [52]. As judged from these results it
is evident that the influence of cell line on embryo development and gene expression lasts as late as until the
elongated stage.
The expression of others trophoblastic markers has been found deregulated in bovine cloned embryos
produce with adult cells. For instance, even when EOMES expression increases significantly from Day 7 to Day17 in
cloned embryos as well in IVF embryos, the expression level of this gene in cloned embryos was significantly lower
than in the IVF counterparts [52]. In a global gene expression assay employing bovine homologous microarray and
using the same embryos as above, 47 genes (3.6%) were found deregulated in cloned embryos. In this study 1321
genes were expressed in the samples out of a 1800 genes represented in the array [52]. Several of those deregulated
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genes were related with trophoblastic functions; this included INFtau, TKDP1 (coding for trophoblast Kunitz domain
protein 1), KRT18, (coding for keratin type-I cytoskeleton protein 18) and KRT8 which were up regulated in cloned
embryos [52]. At least one report correlates the overexpression of KRT8 in the trophoblast of in vivo produced bovine
blastocysts with a high rate of early embryonic loss [90]. The abnormal regulation of the expression of genes related
with trophoblast function and embryo-maternal recognition during peri-implantation would explain the low pregnancy
rate and placentation problems observed in cloned embryos.
Pluripotence markers have been also found aberrantly expressed in the same embryos used in the
experiment mentioned above. While the expression of OCT4 and SOX2 was evident in all cloned embryos tested,
FGF4 and NANOG expression was detected in some but not all cloned embryos. In a quantitative analysis no
significantly difference were found in the expression level of OCT4 in cloned embryos compared with the IVF embryos.
On the contrary FGF4 and NANOG expression was significantly lower in cloned embryos. This an interesting result
since SOX2 expression was not detected in all embryos at blastocyst stage, suggesting that probably only those
blastocysts expressing SOX2 are able to develop further. Similarly, even when OCT4 was properly detected in all
cloned blastocyst, the expression level of this gene was significantly higher in those embryos compared with the IVF
counterparts [52]. One could infer that only those embryos with a normal gene expression of OCT4 will elongate or at
this stage the regulation of this gene is more accurate than in blastocyst [26,52]. Likely only those embryos with
closer gene expression pattern to a “normal stage” will elongated properly. On this sense normal DNA methylation
status has seen reestablished at the time of elongation in cloned bovine embryos [91].
In general only 16% of the tested cloned embryos had a similar expression pattern of the IVF embryos
used as control. This data suggests that at least in some cases (16% in this study), adequate nuclear reprogramming
occurred and persisted through elongation [52]. This proportion is also comparable with the actual rate of live cloned
bovine offspring obtained with these embryos (14%) [52] and with international published reports (10 to 15%) [92-94].
The data presented above concerning gene expression in bovine cloned embryos is in agreement with the
low potential of development of those embryos compared with IVF embryos from the morphological point. The
frequency of successful elongation and of embryonic disc formation was lower in the cloned embryos [52]. The
embryo quality at the time of transfer to recipient cattle has been associated before with the degree of elongation and
gene expression pattern during peri-implantation [16,95].
During elongation of bovine cloned embryos, others genes have been reported to be deregulated such as
Dnmt-1, IGF2 and IGFBP3 (Insulin-like growth factor binding protein 3) [96]. Also at this stage microRNA (miRNA) are
deregulated in bovine cloned embryos produced from an adult cell line [96]. In this experiment fifteen miRNAs were
differentially expressed in the cloned bovine embryos compared with the IVF embryos used as control. From the total
of aberrantly expressed miRNA in cloned embryos, 7 were up regulated while 8 were down regulated. By searching
MIRBase, 7 clusters encompassing 18 miRNAs in 5 bovine chromosomes were identified. Interestingly, miRNAs
within the same cluster showed the same reprogramming pattern. It has been reported that miRNAs play important
regulatory roles during embryo development [98-100]. In this experiment, two miRNAs (miR-30d and miR-26a) that
were fund down regulated in bovine cloned embryos interact with trophoblast Kunitz domain protein (TKDP), which
correlate with the over expression of TKDP in those embryos [52,97]. This result suggest a directly effect of miRNA
deregulation on pregnancy recognition problems seen in bovine cloned embryos.
All the data presented demonstrated that the effect of somatic cell nuclear transfer on gene expression
observed at early blastocyst stage last to the elongated stage affecting the cloned embryo competence. Elongated
embryos are very attractive raw material to understand basic embryo and placental development in bovine embryos.
At present, the methods to obtain elongated bovine embryos are expensive and laborious. The establishment of a
model using small ruminants as goat and sheep to produce elongated bovine embryos seems attractive. For instance
preliminary experiments have been done where elongated cloned and IVF bovine embryos have been obtained
efficiently [101-102].
V. CONCLUSIONS
This review is clear evidence that even thought SCNT is an attractive reproductive technology due to its
potential applications; much is still to be done to improve embryo ability to produce a healthy offspring. The deregulation
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of gene expression patterns during the period prior to implantation is a consequence of an incomplete reprogramming
of the donor nucleus and it is reflected on embryo viability and ultimately on offspring rate. Despite the huge amount
of information gathered about gene expression in cloned embryos its biological relevance is still no clear. Nevertheless,
determining which genes are more susceptible to be deregulated by different protocols of SCNT could be useful in
designing screening strategies to predict embryo competence and success of implantation.
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