Factors and molecules that could impact cell differentiation in the embryo generated by nuclear transfer

ABSTRACT

Somatic cell nuclear transfer is a technique to create an embryo using an enucleated oocyte and a donor nucleus. Nucleus of somatic cells must be reprogrammed in order to participate in normal development within an enucleated egg. Reprogramming refers to the erasing and remodeling of cellular epigenetic marks to a lower differentiation state. Somatic nuclei must be reprogrammed by factors in the oocyte cytoplasm to a rather totipotent state since the reconstructed embryo must initiate embryo development from the one cell stage to term. In embryos reconstructed by nuclear transfer, the donor genetic material must respond to the cytoplasmic environment of the cytoplast and recapitulate this normal developmental process. Enucleation is critically important for cloning efficiency because may affect the ultrastructure of the remaining cytoplast, thus resulting in a decline or destruction of its cellular compartments. Nonetheless, the effects of in vitro culturing are yet to be fully understood. In vitro oocyte maturation can affect the abundance of specific transcripts and are likely to deplete the developmental competence. The epigenetic modifications established during cellular differentiation are a major factor determining this low efficiency as they act as epigenetic barriers restricting reprogramming of somatic nuclei. In this review we discuss some factors that could impact cell differentiation in embryo generated by nuclear transfer.

KEYWORDS:

• enucleation
• cloning
• cell differentiation
• epigenetic
• zygote
• biotechnology
• stem cell

INTRODUCTION

In genetics and developmental biology, somatic cell nuclear transfer (SCNT, i.e., cloning) is a technique in which the nucleus of a cell (called “donor nucleus”) is transferred to the cytoplasm of an enucleated egg (an egg that has had its nucleus removed). Once inside the egg, the “donated nucleus” is reprogrammed by egg cytoplasmic factors to allow embryo development. Somatic cell nuclear transfer can be used in embryonic stem cell (ESC) research or in regenerative medicine, in which it is sometimes referred to as “therapeutic cloning”. It can also be used as the first step in the process of reproductive cloning. Cells of multicellular organisms are functionally heterogeneous owing to the differential expression of genes. Historically, differential gene expression had been thought to involve genetic elimination of those genes that were silenced and retention of others that were expressed in a particular tissue.^1–3

Somatic cells do not have the ability to generate a complete organism and the nucleus of a somatic cell must be reprogrammed in order to participate in normal development within an enucleated egg.⁴ The molecular mechanisms required to stably repress genes are gradually established as embryogenesis and post-embryonic development proceed. Remarkably, the egg and oocyte can reverse this process of repression, disassembling repressive features of nuclear organization and, in particular circumstances, recreating a state of pluripotency (the capacity of a single cell to differentiate into cells from all the three germ layers of the embryo, but not extra-embryonic tissues) or even totipotency (the capacity to form all cells in a new organism, including embryonic and extraembryonic tissues).⁵ It has been known since the earliest experiments that the outcome of nuclear transfer-derived embryo development decreases dramatically as nuclei are taken from increasingly differentiated cells. The differentiated state of cells is very stable, and it is unusual to change fate of cells that have embarked on one pathway of differentiation.⁶

In mammals, the most obvious defects of SCNT-derived embryos are related to the placenta. Commonly observed abnormalities include shorter life span, enlarged placenta, obesity, respiratory problems, and large offspring syndrome.⁴ In most mammalian species studied thus far, the survival rate to birth for cloned blastocysts is only about 1% to 5%, compared with a 30% to 60% birth rate for in vitro fertilized (IVF) blastocysts and developmental and physiological abnormalities have been observed in a significant proportion of the fetuses obtained.^7,8 Because many of these abnormalities are not inherited, it is thought that they are not caused by deficiencies in chromosome replication, but rather by a failure to reprogram epigenetic characteristics of somatic cells, especially imprinted genes.⁹

Epigenetic reprogramming can be defined as any meiotic or mitotic alteration that does not result in a change in DNA sequence but will have a significant impact on the development of the organism.¹⁰ During development of multicellular organisms, different cells and tissues acquire different programs of gene expression. It is substantially regulated by epigenetic modifications such as DNA methylation, histone tail modifications and non-histone proteins that bind to chromatin.¹¹ Thus, each cell type has its own epigenetic signature which reflects genotype, developmental history and environmental influences, and is ultimately reflected in the phenotype of the cell and organism. For most cell types these epigenetic marks become fixed once the cells differentiate or exit the cell cycle.¹²

For successful nuclear transfer and development of the resulting embryo, the nuclear stage of the donor nucleus has to become similar to that of a normal zygotic nucleus. The donor nucleus must adopt the cell cycle parameters of the zygote, including DNA replication, nuclear envelope breakdown, chromosome condensation and segregation, and, subsequently, embryonic patterns of DNA replication and transcription. The cytoplasm of the recipient oocyte, egg or blastomeres has to direct this reprogramming of the donor nucleus.⁵

Nuclear de-differentiation through transplantation of the nucleus into an enucleated oocyte is an experimental approach to reprogram somatic cells. Nuclear transfer provides a powerful tool for studying key aspects of developmental biology and has also numerous potential applications in agriculture and regenerative medicine. SCNT is ultimately aimed at generating undifferentiated stem cells that may be useful for medical research and cell replacement therapies.¹³ Due to the vast literature in the SCNT field, we have limited this review to discuss possible factors and molecules that could impact cell differentiation in the embryo generated by nuclear transfer. Initially, a brief review of the basic laboratory strategies for creating a viable embryo from a somatic cell and a female gamete is provided, to facilitate the understanding of the possible factors and molecules that may affect cell differentiation in SCNT-derived embryo.

SOMATIC CELL NUCLEAR TRANSFER TECHNOLOGY: TECHNICAL ASPECTS

Nuclear transfer is a complex multistep procedure that includes oocyte maturation, cell cycle synchronization of donor cells, enucleation, cell fusion, oocyte activation and embryo culture. However, there are many variations between species in the details of the techniques used to make these changes. In some cases, the transferred nucleus successfully controls development to term of the reconstructed embryo.¹⁴

Oocyte maturation and preparation for SCNT

Cloning mammals by SCNT entails the replacement of oocyte chromosomes with the nucleus of a somatic cell. Recipient cytoplasm and nuclear donor are two essential cellular components in determining the proportion of oocytes developing to the blastocyst stage and the efficiency at which live offspring are produced. Most fully-grown oocytes undergo normal meiotic and cytoplasmic maturation, although only a subset of them will develop to the blastocyst stage. This can be related to the differentiation state of the follicle of origin and differences are not always visible in the oocyte at the ultrastructural level. Origins, cell cycle stage, and specific characteristics of a donor cell line all appear to affect nuclear transfer efficiency with respect to live offspring production.^7,15 Besides that, the cytoplasm of the recipient oocyte, egg or blastomeres has to direct this reprogramming of the donor nucleus.⁵ Thus, the understanding of events leading to cytoplasmic maturation, modification and/or damage may have a direct impact on nuclear reprogramming.

The cytoplasmic factors related with nuclear reprogramming are unclear, but they are powerful. Although extensive genomic studies of oocytes have been conducted in mouse oocytes, in human the accessibility of mature oocytes has been a major barrier to studying oocyte genomics.¹⁶ Several researchers described some information about oocyte transcriptome, but for several reasons (biologicals and technical) they did not show a comprehensive view of oocyte maturation.^17–19

Somatic cell nuclear transfer into enucleated oocytes has shown that, when challenged with a somatic nucleus, the oocyte cytosol will attempt to completely erase the somatic epigenetic phenotype and transform the nucleus to a totipotent state.¹⁶ Recent studies about somatic cell-embryonic stem cells fusion suggested that ESCs retain similar as yet undefined components that can initiate the reprogramming of introduced somatic nucleus to confer pluripotency to the somatic nuclei. Therefore, the cytoplasmic environment of both ESCs and oocytes shares the capacity to reprogram a somatic nucleus.^20–22

Oocyte maturation may be even more important than previously thought. Recently, Adona et al. (2016)²³ showed variations in gene expression between oocytes matured in vivo and in vitro. Several genes have been identified that were up regulated in in vitro maturation, including genes related to transcription complexes, oocyte metabolism and genes associated with chromatin remodeling factors, such as H3 histone, family 3A, retinoblastoma binding protein 4 and 7, and bromodomain adjacent to zinc Finger domain, 1A. Chromatin remodeling is a crucial step in gene expression at this stage and may be directly involved in the epigenetic marks that the receptor nucleus presents. If some of these factors are misplaced into the cytoplasm, then the cytoplasmic maturation of the oocyte could directly interfere with the epigenetic reprogramming of the nucleus.

A major step in this technique is to efficiently produce large batches of enucleated oocytes, which requires considerable micromanipulation skills and expensive equipment.²⁴ However, it is important to mention that the egg's nucleus, known as the germinal vesicle, has broken down prior to ovulation and eggs at this stage do not have a nucleus.²⁵

Enucleation of a recipient oocyte is critically important for cloning efficiency. Enucleation may affect the ultrastructure of the remaining cytoplast, thus resulting in a decline or destruction of its cellular compartments. Even if a transferred nucleus has the potential to support development of a SCNT-derived embryo to the blastocyst stage, meiotic division can be arrested at any stage if metabolism is limited due to destruction of host cytoplasm ultrastructure.²⁶ Some studies suggests that oocytes enucleated at metaphase 2 (M2) stage are better recipients for SCNT when compared to oocytes enucleated at the pro-metaphase 1 stage (8.1% and 53.5%, respectively).^27,28 This in itself imposed additional technical challenges in order to be able to remove the metaphase spindle without causing harm to the remaining cytoplasm, and following transplantation of the nucleus, to induce parthenogenetic activation. There is a great difference between species in the transparency of oocytes, from the relatively clear mouse oocyte to the opaque porcine oocytes.¹⁴

Removing metaphase chromosomes is generally carried out by aspirating the ooplasm adjacent to the first polar body (Pb1) since oocyte metaphase chromosomes are closely adjacent, or attached to, the Pb1. Another method for oocyte enucleation is labeling the oocyte DNA with nuclear fluorescent dyes, such as Hoechst 33342. However, exposing oocytes to UV irradiation may be detrimental to mitochondrial DNA and it must be considered.²⁹ To avoid UV light damage, an alternative is Pb1 enucleation by herniation.^30–33 This procedure, only the extruded cytoplasm is stained with Hoechst 33342 to confirm enucleation and the oocyte is not exposed to UV light. Another fluorescent dye that can be used for oocyte enucleation is Sybr14. Compared to Hoechst 33342, Sybr14 causes less damage to the oocyte due to lower energy transfer and allows higher blastocyst development.²⁹ There are other enucleation protocols to produce recipient cytoplasm for cloning mammals by SCNT such as telophase enucleation,^34–36 chemically assisted enucleation,^26,37–39 enucleation with sucrose pretreatment,⁴⁰ noninvasive enucleation using a pol-scope microscope,^41,42 oocyte bisection,^43,44 enucleation by centrifugation.⁴⁵

Preparation of donor nuclei cells

Since the first reports from Wakayama et al.⁴⁶ about mouse cloning utilizing adult donor cells of female reproductive tissue origin, it is accepted that any cell type of either sex can be used for SCNT. However, the efficiency of SCNT and the clone phenotype vary greatly with regards to the donor cell type used.¹³

The ability of the oocyte to reprogram the donor nucleus and result in successful development is heavily influenced by the donor cell type⁴⁷; the nuclei of cells that are relatively less differentiated support better full-term development compared with those of fully differentiated cells. Advanced and more differentiated cell types have undergone modifications of their cellular structures that restrict their reprogrammability by oocytes. These modifications are of epigenetic origin, since the genetic makeup of the cells is not altered. Also this reveals that the epigenetic state of a donor nucleus restricts the ability of oocytes to fully reprogram the genome after SCNT, and this must be taken into account when new methods for cellular reprogramming are considered.⁴⁸

Experiments in the mouse have shown that donor cells at different stages such as G1, G2 and M phases are compatible with successful cloning,^46,49,50 whereas rapidly cycling cells in S phase are associated with low rates of cleavage. It was shown that co-ordination of the cell cycle between recipient cytoplasm and donor nucleus at the metaphase stage can result in successful cloning using zygote stage mouse embryos as recipients as opposed to eggs arrested in M2.⁵¹ Several methods have been used to control the cell cycle state of the donor nucleus, including serum starvation⁵² and treatment with the microtubule-disrupting agent nocodazole.⁵³

Somatic cell as donor nuclei

Cloned mammals have been produced from various somatic cell types. However, it is still unclear which cell type is the most appropriate.^54,55 Moreover, the differentiation status of somatic cells may have no relationship with cloning efficiency. In 1997, Wilmut and coworkers⁵⁶ demonstrated that viable offspring could be derived from quiescent embryo-, fetal- and adult-derived mammalian cells. In 1998, Wakayama and coworkers⁴⁶ used three different cell types (Sertoli, neuronal and cumulus cells) for SCNT. They found that some enucleated oocytes receiving Sertoli or neuronal nuclei developed in vitro and implanted following transfer, but none developed beyond 8.5 days post coitum. Moreover, a high percentage of enucleated oocytes receiving cumulus nuclei developed in vitro. After embryo transfer, many of these embryos implanted and, although most were subsequently resorbed, some developed to term (2 to 2.8%). According to Mullins and colleagues,²⁸ the low success rate of SCNT, there is a possibility that surviving clones are in fact derived from the nuclei of rare stem cells present in adult tissues, rather than from the nuclei of differentiated cells.

Embryonic stem cell as donor nuclei

Stem cell is capable of self-renewal and generating several differentiated cell types and can be considered as a functional unit of embryogenesis and adult tissue regeneration. Stem cells can be categorized into two types, embryonic and adult, the form capable of generating cells of all three germ layers of the body with indefinite self-renew under certain defined conditions.^57–60

ESCs lines are derived from the inner cell mass (ICM) of embryos at the blastocyst stage, and can be cultured in vitro without becoming aneuploidy. They exhibit developmental pluripotency and can be used to generate chimeric mice on injection into host blastocysts.^28,61 Because rapidly dividing cell populations are asynchronous, some researchers use the cell size as criteria for nucleus donor. Small cells were assumed to be in G1-phase while large cells were considered to be in G2/M-phase. According to Zhou and coworkers,⁶² significantly higher numbers of M2 nuclei developed to the blastocyst stage than interphasic nuclei.

Some studies have found that heterozygosity of the donor cell genome is an important issue in postnatal survival. Eggan and colleagues⁵⁰ compared ESCs cells from inbred and F1 genetic backgrounds and found out that the offspring derived from F1 donor nuclei survived to adulthood, whereas the pups derived from inbred ES cells died shortly after birth. The F1 ES cells have great practical implications for generating animals with multiple genetic alterations without the need to produce chimeric intermediates.

Another point to be considered is about cell culture conditions. ESCs cell confluence dramatically affects the developmental potential of reconstructed embryos. Gao and coworkers⁶³ showed that at 80% confluence, nearly a third of live offspring survived to adulthood. Although at 60 to 70% confluence, only one live pup was born. The same research group also noted that cell passage number can affect the ability of ESCs cell to aggregate with tetraploid embryos, and give rise to viable offspring. According to researchers, at the passage 19, three live pups were born and one reached adulthood. Nevertheless, at passages 22 to 25, no fetuses survived.

Recently, reprogramming human somatic cells into pluripotent ESCs by SCNT has been achieved. Past attempts to produce human NT-ESCs have failed, but Tachibana and colleagues⁶¹ identified premature exit from meiosis in human oocytes and suboptimal activation as key factors are responsible for these outcomes. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic

SCNT-derived embryos in vitro development

During embryo development, the processes of differentiation are controlled by temporal and spatial patterns of gene expression. In embryos reconstructed by nuclear transfer, the donor genetic material must respond to the cytoplasmic environment of the cytoplast and recapitulate this normal developmental process. It is believed that the mRNAs and proteins acquired by the oocyte during its growth and final maturation allow the zygote to go through the early stages of embryo development up until the moment when the embryo produces these factors on its own. The point at which embryonic transcription begins and maternal mRNA is replaced by embryonic mRNA is referred to as the maternal-zygotic transition (MZT). The onset of MZT is species-dependent (for a review, see refs. 64, 65). For instance, zygotic transcriptional activation occurs very early in mouse embryos at the two-cell stage and in ruminants, it is activated at the 4 to 16-cell stage. Thus, mouse SCNT embryos have much shorter window of time in which to reprogram a fused somatic nucleus, and this may result in an incomplete reprogramming in many genomic regions of the donor chromatin, restricting developmental potential.⁴⁸ The MZT is thought to be an important and limiting step of development because it coincides with a developmental block observed in embryos cultured in vitro.

According to Brambrink and colleagues,⁶⁶ the transcription profile of ESCs derived from embryos resulting from a normal fertilization or SCNT are identical, and it has been suggested that only nuclei that have undergone appropriate epigenetic reprogramming are capable of generating ESCs from SCNT embryos. Furthermore, full-term development of mammalian embryos also requires and spatial organization of cells and failure in these events can lead to developmental abnormalities.⁴⁸ One of the first spatial events during embryo development is the formation of the trophectoderm (TE) which gives rise to extraembryonic structures; there may be the major impediment to the generation of adult animals from SCNT.

REPROGRAMMING GENE EXPRESSION

Reprogramming refers to the erasing and remodeling of cellular epigenetic marks to a lower differentiation state, such as fertilization during mammalian development. Complete nuclear reprogramming restores the condition of totipotency, which is the capacity to generate all kinds of cells, including extraembryonic cells, enabling the SCNT clone to develop into a normal adult. The persistence of epigenetic marks on the somatic genome makes crucial developmental genes inaccessible for transcription, leading in some cases to abnormal gene expression patterns. There are however important differences between species.^67–69 The most effective reprogramming methods, SCNT and induced pluripotent stem cells (iPSCs), are widely used in biological research and regenerative medicine, yet the mechanism that reprograms somatic cells to totipotency remains unclear and thus reprogramming efficiency is still low.⁷⁰

The pool of oocyte messenger RNA sustains a highly program of gene expression essential for its own development, maturation, and fertilization as well as for early embryonic development. Before maturation, oocytes are transcriptionally active. Transcription stops when oocytes undergo meiosis to form eggs. After germinal vesicle breakdown, gene expression is mainly under post-transcriptional control, which involves differential degradation, stabilization and storage of transcripts, and their timely recruitment to the translation machinery. Following fertilization the early embryo is essentially transcriptionally silent and the early development is directed by the complement of maternally inherited mRNAs and proteins.^71,72

Somatic nuclei can be reprogrammed to a pluripotent state by factors in the oocyte cytoplasm, and that reprogrammed nuclei can drive embryonic development to term. The ooplasm from different species may have different capacity to demethylate specific genes.⁷³ The cytoplasm of bovine, porcine, and rabbit oocytes has been shown to be able to initiate the first steps of the reprogramming process after SCNT in donor cells of different species. However, development ceases early in embryonic development, indicating a high degree of species specificity of the epigenetic reprogramming process and that successful interspecies SCNT is unlikely to succeed.⁷⁴ Failure to achieve development to term has generally been attributed to a number of reasons, these include: (a) technical limitations in embryo reconstruction, (b) the use of incompetent or low quality oocytes and (c) the lack of cell-cycle synchronization between the donor and recipient cells.⁴⁸ Cell fusion experiments demonstrate that reprogramming factors are dominant and that when a less-differentiated cell is fused with a more-differentiated one, the resulting phenotype is that of the less-differentiated one.⁷⁵

Cibelli and coworkers⁷⁵ compared the gene expression profile of blastocysts derived from two different cell lines (i.e., one cell line produced live offspring and the other failed to do so). They found out that of the 14806 probe sets analyzed, 3077 transcripts were differentially expressed. Also, embryos derived from these two cell lines showed a very similar expression profile to that of IVF-derived embryos, indicating that a major degree of reprogramming took place by the time the embryos reached the blastocyst stage. Only 212 probe sets were differentially expressed between embryos derived from the high-efficiency and low-efficiency cell lines. Interestingly, a set of genes maintained their relative levels of expression in both the cell lines and the SCNT blastocyst, probably representing genes that failed to be reprogrammed by the oocyte. These genes were related to biological functions such as cell morphology, posttranslational modifications, gene expression, cell cycle, carbohydrate metabolism, and cell signaling.

Nuclear reprogramming after SCNT may be divided into two major events. The first is a reversal of the program of pluripotency; and the second step is the establishment of new differentiation programs.^15,48 The first event leads to timely reactivation of a zygotic program, involving the correct reactivation of embryonic genes and repression of somatic genes. The second event is the initiation of the differentiation program that commences when the TE arises as the first lineage in the embryo.

Octamer-binding transcription factor 4 (Oct-4) has been used as a marker for reprogramming in many studies that use ES cells or oocytes as a reprogramming environment. Oct-4 is expressed in the mouse ICM and in the ICM and TE of pig and cattle embryos, and is continuously required to sustain pluripotency in mouse.⁷⁶ However, Oct-4 is detected at very low levels in mesenchymal stem cells suggesting that this gene cannot be used as a unique indicator of multipotency. Another gene called Nanog (a homeobox containing transcription factor) has also been described as a maker of nuclear reprogramming process. According to Chambers and coworkers,⁷⁷ Nanog is expressed in ESCs cells and is essential for the maintenance of pluripotency and self-renewal.

Wakayama¹⁵ examined the TE and ICM lineages using Cdx2 (a caudal type homeobox 2) and Oct-4 that are key genes for the specification of TE and ICM, respectively. He found out that blastocysts expressing Oct-4 were lower as 50%; however, regardless of Oct-4 expression, more than 90% of the cloned embryos expressed Cdx2, suggesting that those embryos have a reduced potential to produce the ICM lineage, the TE lineage can be established and maintained.

Induced pluripotent stem cells – iPSCs

iPSCs is another method to reprogram somatic cells in vitro. The study of the nuclear reprogramming processes of iPSCs has helped to clarify many aspects of reprogramming in nuclear transference. Thus, comparisons between these two techniques of reprogramming are justified. Induced pluripotent stem cells can be generated directly from adult cells that have been reprogrammed to a type of pluripotent stem cell via the exogenous expression of transcription factors. The most well-known type of pluripotent stem cell is the embryonic stem cell. Unlike ESCs, they bypass the need for embryos or other ethically charged cells; they are autologous and can be generated from individuals of any age.⁶⁰ Additionally, iPSCs provide an attractive alternative to ESCs since they reduce or avoid immune rejection by generating stem cells from the patient´s own cells.⁷⁸ Thus, iPSCs could be a promise for disease modeling, drug selection and cell therapies in both regenerative medicine and agriculture.⁷⁹

The generation of iPSCs is slow and inefficient comparing with SCNT, but iPSCs are much easier to obtain. The iPSCs are commonly derived from somatic cells by transfecting two pluripotent transcription factors, Oct4 and Sox2 (sex determining region Y box 2), and two protooncogenes, c-Myc (v-Myc myelocytomatosis avian viral oncogene homolog) and Klf4 (Krueppel-like factor 4).⁸⁰ The combinations of transcription factors, additions of small molecule, and culture conditions have been modified to improve the efficiency of iPSC derivation. All conditions aim to globally reset the epigenetic and transcriptional state of somatic cells into that of pluripotent cells. This technique produces alternative pluripotent cells that closely resemble blastocyst-derived ESCs. However, the main defects of iPSCs are tumorigenicity and low efficiency,^70,81–83 which restrict the use of iPSCs in clinical applications.

For iPSCs, in recent review, David and Polo⁸⁴ showed the nuclear reprogram in threes moments: the initiation, maturation and stabilization phases. The initiation phase was defined as the commencement of the reprogramming process until the first pluripotency-associated genes were expressed. This is characterized by a loss of the somatic cell signature (such as the loss of the transcription factors Snai1/2 or Zeb1/2) and the gain of an epithelial signature, such as the expression of Cdh1 or EpCam. The maturation phase is characterized by the second wave of major transcriptional changes and is marked by the onset of the first pluripotency-associated genes (Fbxo15, Sall4, Oct4, followed by Nanog and Esrrb). In the end of this phase it is possible to detect Sox2 or Dppa4 too. The stabilization phase is characterized by changes that occur in iPSCs after they have acquired pluripotency, with the expression of Sox2, Dppa4, Pecam.

NUCLEAR REPROGRAMMING AND EPIGENETIC CONTROL

Normal development depends on a precise sequence of changes in the chromatin structure, which are primarily related to the acetylation and methylation status of histones and the methylation of genomic DNA.⁸⁵ The process of returning a differentiated somatic nucleus to a totipotent state is termed nuclear reprogramming. Reprogramming is likely to have a crucial role in establishing nuclear totipotency in normal development and in cloned animals, and in the erasure of acquired epigenetic information. During normal early mammalian development, reprogramming of genomic DNA modifications is observed shortly before and after the formation of the zygote.⁸⁵ The embryonic DNA is increasingly remethylated between the two-cell and the blastocyst stages,⁸⁶ which concur with the specie-specific onset of MZT.⁸⁷ These mechanisms ensure the normal early development. However the application of assisted reproductive technologies may induce aberrant mRNA expression patterns in the resulting embryos and greater epigenetic disturbances.⁸⁵

Somatic cell nuclear reprogramming involves dedifferentiation of the already differentiated donor cell to a totipotent state followed by a redifferentiation of SCNT embryo to different precursor and somatic cell types during later development. During these steps may occur normal and abnormal reprogramming, what may explain many of the defects in SCNT embryos that happen in the later reprogramming stage and correlate to placenta development and function alterations.⁸⁸

The genetic information (encoded in the DNA sequence) of all cells in a multicellular organism is almost invariable. Despite this, cells have the potential to differentiate into distinct cell types with unique cellular programs, morphologies, and functions. This outstanding feat is achieved by epigenetic mechanisms, that includes (1) DNA modification (DNA methylation), (2) post-translational histone modification by methylation, acetylation phosphorylation or ubiquitination, (3) chromatin remodeling by altering the location of nucleosomes, (4) small noncoding RNAs that regulate gene expression at the post-transcriptional level that determine cell fate.^89,90 Combination of these modifications characterizes the chromatin configuration and the accessibility of genes to the transcription machinery and consequently, transcriptional regulation of the expression of genes. The timing and manner to achieve an embryonic-like chromatin structure conformation will depend on the type of donor cell used. Embryonic stem 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 ESC more accessible to the cytoplasm of the recipient oocyte and to efficient reprogramming.⁹¹ However, the epigenetic mechanisms that are responsible for the transformation from a differentiated somatic cell into a pluripotent state remain unknown.⁹²

Epigenetic mechanisms include, but are not limited to, DNA methylation (predominantly at CpG dinucleotides), post-translational modifications of histones, non-coding RNAs (ncRNAs) and chromatin remodelling.⁹³ DNA methylation and histone modification are the main epigenetic modifications of the genome that regulates crucial aspects of its function. By the blastocyst stage there is a marked increase in methylation of DNA and histones, predominantly in the ICM, indicating epigenetic differences between the ICM and TE lineages.⁹⁴ The epigenetic mechanisms alter chromatin in such a way that changes the availability of genes to transcription factors required for their expression. These alterations are assigned to DNA methyltransferases (they add a methyl group to cytosine in the dinucleotide CPG), Polycomb Group (PcG) and associated proteins, which modify histones.⁹⁵

DNA methylation in SCNT embryos

Genomic methylation patterns in somatic differentiated cells are generally stable. It involves the addition of a methyl group to the 5′ position of the cytosine pyrimidine ring to generate 5-methylcytosine (5^mC) and these methylated CpGs play a central role in transcriptional regulation in mammals. DNA methylation is a heritable epigenetic marker by which expression of a gene may be regulated through alteration in local chromatin structure and in most situations, is associated with the repression of transcriptional activity.^92,93,96 However, in mammals there are at least two developmental periods (germ cells and preimplantation embryos) in which methylation patterns are reprogrammed, generating cells with a broad developmental potential.¹¹² Very recently, mammalian genomes have been shown to also possess adenosine methylation, although the physiological consequence of this remains unclear. Nevertheless, modifications involving DNA methylation and alkylation damage of nucleic acids are tightly linked with many diseases.^93,97

The most intense epigenetic reprogramming occurs in SCNT cloning when the expression profile of a differentiated cell is abolished and the new embryo-specific expression profile is established to drive embryonic and fetus development. It is estimated that this includes an abrogation of the expression of 8000 to 10000 genes of the somatic cell program and the initiation of the embryonic program with about 10000 genes. Pre-zygotic reprogramming includes the erasure of somatic cell epigenetic modifications and is followed by a post-zygotic establishment of embryonic modifications. Other post-zygotic reprogramming includes X-chromosome inactivation and adjustment of telomere lengths.^98,99

DNA methylation reprogramming in early embryos is regulated by methylation and demethylation related genes. The paternal DNA is actively and rapidly demethylated after fertilization, while the maternal DNA undergoes passive demethylation. The embryonic DNA is increasingly remethylated especially from eight-cell to blastocyst stages.^98,100 According to Dean et al. (2003),¹⁰¹ while in normal bovine embryos the demethylation of the maternal and paternal genome is complete, and de novo methylation occurs at the eight-to-sixteen-cell stage, in cloned embryos, demethylation is not complete and de novo methylation occurs at the four-cell stage. The biological significance of early embryo demethylation is uncertain, but it seems to be necessary to remove differences in gamete-specific methylation patterns and then to revise the genome before normal development.

DNA methylation mainly depends on the activity of a class of enzymes, the DNA-methyltransferases (DNMTs) and removed via a pathway involving specific enzymes, for example ten-eleven translocation methylcytosine dioxygenase 1 (TET1) which catalyses conversion of 5^mC to 5-hydroxymethylcytosine (5^hmC).⁹³ The DNMTs modulate the expression of genes (especially imprinted ones) as well as X chromosome inactivation. There are five main DNMTs that are important in maintenance DNA methylation: DNMT1, DNMT10, DNMT3a, DNMT3b and DNMT3L.⁹² During early development, DNA methylation is stated by DNMT3a and DNMT3b and maintained by DNMT1 during DNA replication.^102,103 The oocyte-specific isoform, DNMT1o, maintains maternal imprints. DNMT 3L co-localizes with DNMT 3a and -b and presumably is involved in establishing specific methylation imprints in the female germline.¹⁰⁴

Cloned embryos have aberrant DNA methylation that are likely associated with suboptimal demethylation events compared to adequate timed parental genome demethylation after fertilization.⁸⁶ It is generally believed that low cloning efficiency is mainly due to incomplete epigenetic reprogramming.^89,98,100 Interestingly but not surprising, epigenetic marks examined in cloned embryos and adults from several species show abnormalities and most SCNT embryos also differ from each other in their precise epigenetic profile.¹⁰¹

Studies comparing the changes in DNA methylation patterns in cell nuclei showed differences in cells of normal embryos when compared with SCNT embryos.^86,105 These authors revealed that the nuclear pattern in the SCNT embryos better resembled that of the somatic donor cells used for cloning, leading to the conclusion that nuclear reprogramming was incomplete and the epigenetic modifications of differentiated donor cells was not erased and returned to the totipotent state of early zygotic cells.

In cloned bovine morulae and blastocysts, methylation levels of several repeat and unique sequences were found by bisulfite analysis to be much higher than in normal embryos, and thus resembled methylation levels in the donor-cell genome.^86,106 SCNT-derived embryos in pig revealed efficient demethylation of all sequences analyzed whereas the development to term of pigs produced by SCNT is no higher than that of the cow.¹⁰⁷ Other erroneous epigenetic patterns have been observed in cloned embryos such as hypermethylation of trophoblast DNA at the blastocyst stage and increased levels of histone H3K9 methylation.^8,108 However, it is not yet possible to conclude whether the altered methylation levels caused the abnormalities and developmental failure or whether abnormal development has led subsequently to faulty DNA methylation.⁸⁵

Histone modifications in SCNT embryos

Chromatin structure is crucial for gene expression. DNA in eukaryotic cells is compacted and packaged into a macromolecular complex termed chromatin, the fundamental unit of which is a nucleosome. Nucleosomes consist of a histone protein octamer (2 each of histones H3, H4, H2A and H2B) around which approximately 1.75 turns of DNA are wound. Within this setting, histones are subject to many post-translational modifications that have the potential to encode epigenetic information.⁹³

Euchromatin (lightly packed form of chromatin) is a predominant structure allowing gene accessibility and reprogramming to pluripotency. Therefore, it is not surprising that histone modifications may influence the global gene expression by altering chromatin structure. Histones N-terminal tails can be modified by acetylation, phosphorylation, ubiquitination or methylation and these alterations play an important role in the regulation of gene expression.^88,92 Still, histone acetylation and methylation are the most commonly studied modifications of histones tails.

Histone acetylation is the main type of histone modification during oogenesis and it is crucial in epigenetic reprogramming. Histone acetylations causes decreased interaction between the histone and DNA and is generally associated with active transcription.⁹⁶ The level of histone acetylation may be correlated with the regulation of gene expression since the higher the histone acetylation, the greater the expression of a given gene.¹⁰⁹ Still, histones can also be deacetylated and this modification is catalyzed by histone deacetylases (HDACs). Histone deacetylation is associated with a closed chromatin structure and gene repression.⁹⁵

Histones can also be modified by histone methyltransferases (HMTs). These enzymes are able to methylate histones at lysine residues and are associated with both gene activation and repression, depending on which lysine is methylated and whether it is mono-, di-, or tri-methylated. In general, trimethylation of histone H3 on the lysine at the 4^th amino acid (H3K4me3), H3K36me, or H3K79me, which are known markers of active transcriptional activity, are localized to euchromatin while H3K9me2/3, H4K20me3, and H3K27me3 are markers of transcriptional repression and are localized on heterochromatin.¹¹⁰ Proteins from PcG are also able to modify histones by methylation; they form polycomb repressive complexes that are able to modify chromatin structure to maintain gene silencing.⁹⁵ For example, PRC2, a polycomb repressive complex required for embryonic development in mammals, catalyzes the methylation of histone H3 lysine-27 (H3K27), what leads to additional repressive complexes recruitment such as PCR1 that adds an additional level of repression that is propagated.^111,112

Inducing epigenetic alterations in vitro

Because the epigenome regulates global patterns of gene expression that define cell function, and because SCNT requires a global change in gene expression from a functional to an embryonic pattern, it is not surprising that cloned embryos have altered methylation. The majority of researches on SCNT describe efforts to overcome the inefficiencies of the process itself. These studies include attempts to identify the best source and treatment of donor cells, improved oocyte activation protocols, and methods of chemically assisted reprogramming. In the latter case, either donor cells before SCNT or the reconstructed embryo afterward are exposed to chemicals thought to either stimulate or inhibit various enzymes involved in remodeling chromatin.¹¹³

The prevention of epigenetic errors such as hypermethylation of DNA may provide an increase in the efficiency of cloning in animals. One of them is a DNA methyltransferase inhibitor called 5-aza-2´-deoxycytidine (5-aza-dC). Using 5-aza-dC in bovine donor cells Enright and colleagues¹¹⁴ increased the blastocyst rate when compared to untreated controls. On the other hand, in pigs, Huan et al. (2013)¹¹⁵ only observed a beneficial effect of 5-aza-dC treatment on cloned embryos but not on donor cells, probably because donor cells do not have some proteins such as PGC7/Dppa3/Stella, which protect imprinted genes, leading to imprinting errors.

Considering that nuclear remodeling in clones is often not complete strategies that facilitate the chromatin opening may be interesting to improve reprogramming efficiency. One of them is a histone deacetylase inhibitor named trichostatin A (TSA). Jeong et al.¹¹⁶ found that TSA treatment improved porcine blastocyst formation rates (to 22%) compared with 8.9% in the non-treatment group. These improvements on the blastocyst rate using TSA could be related to the correct number of cells at the blastocyst stage expressing Oct-4, since it is an important factor for pluripotency maintenance.^117,118 Still, Wang and coworkers¹¹⁹ found that treatment of both donor cells and early cloned embryos with combination of 5-aza-dC and TSA significantly improved the in vitro and full-term development of nuclear transferred bovine embryos.

Treatment also can be done for SCNT embryos. Epigenetic modifier such as TSA seems to improve histone acetylation levels making them more similar to those of IVF embryos than those cloned embryos not treated.¹²⁰ Another epigenetic modifier that can be used to treat reconstructed embryos is the histone deacetylase inhibitors (HDACi) which increases histone acetylation. Because the reprogramming of nuclei following SCNT only happens during a limited time before zygote genome activation, the relaxation of chromatin structure by histone acetylation might facilitate successful remodeling and it can be achieved by using HDACi after the activation of the SCNT embryo.⁹⁶ The mechanism of improvement in cloning efficiency by HDACi may be due to their ability to stimulate nascent mRNA synthesis after increased histone acetylation.¹²¹ Other researchers believe that HDACi supports global chromatin reprogramming and thereby gene expression in several species by acting not only on acetylation of histones but also on trimethylation of H3K9 (H3K9me3) or even DNA methylation.^122–124

Valproic acid (VPA) is an example of HDACi used for cloned embryo treatment that is able to improve development competence of cloned embryos in pigs¹²⁵ and cattle¹²⁶; although the effect of VPA varies among species. In this regard, most factors that promote chromatin decondensation, including histone deacetylase inhibitors, have been found to increase nuclear reprogramming efficiency. Sangalli and coworkers¹²⁷ used VPA in SCNT to investigate whether the treatment of nuclear donor cells with HDACi could improve pre- and post-implantation development of cloned cattle. Their results showed that the treatment of fibroblasts with VPA increased histone acetylation without affecting DNA methylation. On the other hand, in spite of the alterations in fibroblasts resulting from the treatment with VPA, their use as donor cells in SCNT did not improve pre and post-implantation development of cloned cattle.

EFFECTS OF IN VITRO CULTURE (IVC) CONDITIONS ON SCNT-DERIVED EMBRYOS

It is generally accepted that mammalian preimplantation embryos are sensitive to their environment and that conditions of culture can affect future growth and developmental potential both pre and postnatally. The period of post-fertilization embryo culture is the most critical period affecting blastocyst quality assessed in terms of gene expression pattern and ability to establish a pregnancy. Extended IVC of mammalian embryos per se is known to result in aberrations in mRNA expression patterns, affecting imprinted and non-imprinted genes and may also be associated with epigenetic errors.^128,129 In the case of cloning, it is difficult to discriminate between the effects of IVC and dysregulation due to the cloning process. The “large offspring syndrome” that was found in a certain proportion of cloned offspring, was sometimes also observed, although in a less severe form, in offspring derived from IVP embryos cultured under certain conditions.^85,130

The post-fertilization culture environment includes not only the culture medium but also the number of embryos cultured together, the embryo: medium ratio, the temperature and gas atmosphere.¹³¹ According to Rizos and colleagues¹³² addition of serum to culture medium increases the speed of development manifested in a lack of normal morula compaction and the earlier appearance of blastocysts.

Gene expression has a fundamental role in the coordination of homeostatic and metabolic mechanisms throughout life. Precise control of gene expression during the preimplantation phase of development is particularly important and could be impact on SCNT. Rizos et al.¹³³ evaluated embryonic gene expression known to be involved in important developmental processes including apoptosis (Bax), gap junction formation (Cx43) and differentiation (LIF and LIF-Rβ). They found an elevated abundance of transcripts for Bax, as well as reduced expression of the gap junction gene, Cx43.

Although the amount of data on expression of specific genes in both in vitro production (IVP) and cloned embryos has increased significantly, a more complete understanding of the relationship between altered phenotypes and alterations in mRNA or protein expression profiles in IVP and cloned pregnancies is needed. According to Farin and coworkers,¹³⁴ a major difficulty in studying the effects of either specific pre-implantation culture environments or particular cloning methodologies on patterns of gene expression is the lack of connection between alterations in gene expression detected at one stage of pre-implantation development and subsequent effects on developmental competence during the peri- or post-implantation periods.

Improvements to in vitro culture can assist in the production of developmentally competent SCNT-derived embryos, and can thus increase the practical application of blastocyst transfers. However, references on the transfer of SCNT-derived blastocysts are more limited because SCNT-derived embryos are considered more fragile compared to in vitro fertilized (IVF) embryos.¹³⁵

Most SCNT-derived embryos are typically cultured in vitro and transferred at the blastocyst stage. Perturbations in the environment of the preimplantation embryo can modify the phenotype of the resultant offspring in postnatal life. The environmental conditions to which the SCNT embryos are exposed also influence the epigenome and can compensate for a certain level of genetic reprogramming errors. According to Chesné et al. (2002)¹³⁶ and Dinnyes et al. (2008)⁸⁸ embryo transfer in early-stage and asynchrony between the SCNT embryo age and the post-ovulatory age of the recipient oviduct/uterus may reduce aberrations due to IVC conditions. Among the maternal environmental factors that can alter postnatal phenotype are: a low-protein diet, reduced feed intake, induction of inflammation through injection of lipopolysaccharide (LPS).¹³⁷ In addition, the type of culture medium used to support development has been reported to cause changes in the developmental program, as demonstrated by addition of serum to culture medium of mouse embryos.¹³⁸ Some postnatal changes can be considered to reduce offspring fitness while others could enhance fitness under certain environments.

Several molecules produced by the oviduct and endometrium termed “embryokines” regulate embryonic growth and development. This class of molecules includes: protein hormones, growth factors, cytokines, amino acids, prostacyclin 1 and endogenous cannabinoids, which act through various receptors to regulate development and implantation. Alterations in developmental processes caused by embryo culture could reflect, at least in part, the absence of critical maternal regulatory molecules or, for the case of addition of serum to culture medium, alteration of embryonic function by bioactive molecules not normally involved in control of development^137,138

Colony-stimulating factor 2 (CSF2), also called granulocyte-macrophage colony-stimulating factor, is an “embryokine” that has been implicated in developmental programming. In mice, as shown by Sjöblom and colleagues,¹³⁹ addition of CSF2 to culture medium either partially or completely prevented the effect of culture on postnatal growth in females and males, relative brain mass in males and placental weight of female progeny when they themselves became pregnant. However, this molecule did not prevent the effect of culture on fatness of males in the postnatal period. In bovine, addition of CSF2 to culture medium has been reported to increase the proportion of embryos that develop to the blastocyst stage in culture. Addition of CSF2 at day 5 after insemination can increase the percent of embryos that become blastocysts at day 7 after insemination. Moreover, blastocysts that develop following CSF2 treatment had a tendency for more cells in the ICM and a greater ratio of ICM to trophectoderm cells.¹⁴⁰

It is believed that CSF2 acts to increase the expression of genes regulating mesoderm formation and epithelial to mesenchymal transition and decrease the expression of genes regulating neural cell differentiation. An additional effect of CSF2 is related to lower genes involved in apoptosis and increase genes that regulate cell survival. Likely, CSF2 may induce changes in cell fate and survival resulting in an embryo with enhanced competence for survival during the embryonic and fetal periods.^141,142

In addition to “embryokines”, developmental programming in culture could involve changes in cellular energy metabolism in the developing embryo. According to Banrezes and coworkers¹⁴³ alterations in redox potential and mitochondrial activity of the mouse zygote caused by starvation, inclusion of pyruvate or lactate in the culture medium, or raising external pH can result in changes in body weight in the postnatal period. This could certainly impact embryos reconstituted by SCNT.

ABNORMALITIES IN DEVELOPMENT ARISING FROM NUCLEAR TRANSFER TECHNIQUE

Nuclear transfer-related anomalies may be related to several factors such as: inappropriate donor cell and/or recipient oocyte; inappropriate synchrony between the cell cycle phase of donor nucleus and recipient cytoplasm; inadequate reprogramming of the donor genome; inappropriate handling of oocytes, somatic cells and embryos during maturation, various manipulations and cultural techniques causing mechanical, osmotic, electrical, toxic, thermal and other types of damage.¹⁴⁴

Abnormalities in development arising from cloning system are not limited to in vitro manipulation. Maternal diet can impact preimplantation phenotype and long-term development.¹³¹ Some studies have demonstrated that high protein diets in sheep have been associated with reduced viability and increased fetal and birth weights, alterations in fetal development and physiology.^145,146

Several researchers have described various abnormalities that include circulatory distress, placenta edema, hydrallantois, and chronic pulmonary hypertension.^4,147 According to these authors, such abnormalities lead to death shortly after birth. In addition, the actual cloning system shows a high rate of abortion that may be related to a specific deficiency (or combined deficiencies) in the in vitro culture system or the cloning technique itself, leading to incomplete nuclear reprogramming of donor cell.¹⁴⁸ Koo and colleagues¹⁴⁹ demonstrated that cloned blastocysts showed a significantly higher proportion of ICM cells when compared to in vitro- or in vivo-derived embryos. These results indicate that some structural abnormalities, which can affect the survival of cloned embryos, may arise during pre-implantation period.

When the offspring survived they showed large placenta and increased birth weights (large offspring syndrome – LOS), immune dysfunction or kidney/brain malformation which led to death later.^4,150,151 The occurrence of LOS seems to be species-specific; it is quite frequent in cattle, sheep and mice, but almost no malformations were detected in pigs and goats. The LOS is a typical phenotype in cloned mammalian species, but factors responsible for LOS still remain unknown.

According to Vajta and Gjerris,¹⁴⁴ LOS can be described as a number of malformations and diseases that may include: placental abnormalities, fetal overgrowth; prolonged gestation; stillbirth; hypoxia; respiratory failure and circulatory problems; lack of post-natal vigour; increased body temperature at birth; malformations in the urogenital tract (hydronephrosis, testicular hypoplasia); malformations in liver and brain; immune dysfunction, lymphoid hypoplasia, anaemia, thymic atrophy; and bacterial and viral infections.

According to Chavatte-Palmer and colleagues¹⁵² there are two categories of loss after birth: those in the early neonatal period (within 1 week) and those in the following phase lasting up to several months. Early death is caused by the failure of the cardiovascular system and the renal system, sometimes because of internal bleeding, skeletal deformations, gastrointestinal problems and multiple organ failure. Hydrocephalus and infections were also reported in this period. It seems that initial problems are related to events that took place during pregnancy or defects at placentation. Later, the main cause is thymic aplasia, the increased size of pituitary. Some animals die without any real pathological finding, others with diseases that cannot be directly connected with the death, including, for example, liver steatosis. During this period some laboratory findings may be abnormal, but these return to normal later. After 6 months, cloned cattle do not display major differences from control animals.¹⁴⁴

Another problem that may be considered is about the age of cloned animal. The most famous example of this is the cloned sheep Dolly. At the end of the chromosomes there are stabilizers known as telomeres. They shorten a little every time the cell replicates itself; for this reason telomeres are considered as a kind of biological clock in the cells. At present we possess very limited data on the life-span of clones. The importance of telomere length to the survival and health of cloned animals require further research.¹⁴⁴

One other alteration that can occur in cloned embryo is related to X chromosome inactivation (XCI). During the evolution of mammals, two morphologically different sex chromosomes have been generated from a pair of autosomes. In female mammals, the dosage difference in X-linked genes between XX females and XY males is compensated for by inactivating one of the two X chromosomes during early development. The current knowledge on XCI during early embryogenesis mostly comes from studies in mice, which is partly due to the fact that it takes place at a very early phase of mammalian development when the embryo is accessible.¹⁵³ During mouse development, two types of XCI in female cells ensure the silencing of one of the two X chromosomes in a stage-specific manner. Imprinted XCI, which preferentially inactivates the paternal X, is maintained through the preimplantation stage and is inherited in the placental tissues. By contrast, another type of XCI, the so-called random XCI, occurs in cells of the embryo proper after implantation and persists throughout life. Both forms of XCI are triggered by Xist (X-inactivation specific transcript), a noncoding RNA that acts on the future inactivated X chromosome.¹⁵⁴

Many studies have shown that Xist is aberrantly expressed in cloned mammalian embryos or in animals generated by SCNT. Inoue and coworkers¹⁵⁵ reported that Xist was ectopically expressed from the active X (Xa) chromosome in cloned mouse embryos of both sexes. Deletion of Xist on Xa showed normal global gene expression and resulted in about an 8-9-fold increase in cloning efficiency. They also identified a Xist-independent mechanism that specifically down-regulated a subset of X-linked genes through somatic-type repressive histone blocks. Furthermore, Matoba and colleagues¹⁵⁶ described that inhibition of ectopic Xist expression in early cloned male mouse embryos by RNA interference (RNAi) also increased the SCNT efficiency by more than 10-fold. More recently, it has been demonstrated that Xist mRNA expression at the blastocyst, fetal and postnatal stages in cloned pigs is higher than that in vivo fertilization derived pigs.^157–159 Zeng and coworkers¹⁶⁰ demonstrated that injection of anti-Xist siRNA (small interfering RNA) into pig SCNT embryos at the 1-cell stage effectively inhibits Xist expression through the 16-cell stage, which significantly increases the number of cells per blastocyst and improves the birth rate of healthy cloned piglets.

CONCLUSIONS

Although a number of questions regarding the low efficiency of SCNT still remain unanswered, the central role of nuclear reprogramming on the outcome of cloning is evident. Somatic cell nuclear transfer extensively alters the gene expression of differentiated somatic cells. However, a combination of in vitro culture conditions, aggressive manipulation and insufficient cell reprogramming, compromises the developmental potential of SCNT embryos.

Nuclear transfer is a delicate mechanism that can undergo drastic influences by the way the nucleus donor and / or receptor cytoplasm have been manipulated. Cloned embryos present varying degrees of aberrations in chromatin structure and DNA methylation, which cause inadequate expression of developmental genes or the expression of unnecessary somatic genes. In this sense, epigenetic factors seem to play a crucial role in this process. Herein it was reviewed some mechanisms of epigenetic reprogramming, and further discussed experimental methods that could drive to undesirable phenotypes. It has been reported that in vitro culture systems could lead to epigenetic disturbances in embryos and offspring. Cloned embryos and animals are more prone to epigenome abnormalities probably due to the persistence of epigenetic memory of somatic cells impairing the regain of pluripotency. Since that some epigenetic errors result from handling gametes and early embryos in vitro, particularly with cloning by SCNT, strategies to mitigate epigenetic errors such as chromatin modifying agents or factors that could interfere in methylation / acetylation processes may be a viable strategy to increase the efficiency of this technology. Increasing the efficiency of SCNT would have a great impact on biomedical sciences and agriculture. Understanding the reprogramming process of SCNT derived embryos would be crucial to increase the success rate of cloning. More efforts in this field are required to dissipate this technology in every sector of livestock for all species.

The SCNT also represents a moment of extraordinary importance for cell differentiation. During the initial period of the reconstituted embryo, nuclear reprogramming and activation of genes linked to totipotency and pluripotency occur. This unique moment in cell life may have important responses to increase the efficiency of results in sciences such as cell therapy and tissue engineering. Thus, the best understanding of nuclear reprogramming may have an important impact in the basic biomedical sciences and also in regenerative medicine. Apparently, we still have a long way to go.

DISCLOSURE STATEMENT

The authors report no conflict of interest.