Int. J. Dev. Biol. 63: 123-130 (2019)
`https://doi.org/10.1387/ijdb.180324mc
`
`www.intjdevbiol.com
`
`
`Somatic cell nuclear transfer: failures, successes
`and the challenges ahead
`MARTA CZERNIK1,2, DEBORA A. ANZALONE1, LUCA PALAZZESE1, MAMI OIKAWA#,3 and PASQUALINO LOI*,1
`1 Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy, 2 Department of Experimental Embryology, Institute of
`Genetics and Animal Breeding of the Polish Academy of Sciences, Jastrzębiec, Poland and 3 Center for Genetic Analysis of
`Behavior, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Japan
`
`ABSTRACT Somatic cell nuclear transfer (SCNT) has a broad spectrum of potential applications,
`including rescue of endangered species, production of transgenic animals, drug production, and
`regenerative medicine. Unfortunately, the efficiency of SCNT is still disappointingly low. Many fac-
`tors affecting cloning procedures have been described in several previous reviews; here we review
`the most effective improvements in SCNT, with a special emphasis on the effect of mitochondrial
`defects on SCNT embryo/ foetus development, an issue never touched upon before.
`
`KEY WORDS: somatic cell nuclear transfer, SCNT, nuclear reprogramming, mitochondria
`
`Introduction
`The latest Red List of the International Union for Conservation
`of Nature (IUCN) shows that 21% of all mammals as threatened.
`The worst news is that the global number is likely underestimated
`due to lack of recent data from more than 58% of breeds, therefore
`classified as unknown risk (FAO, 2018).
`To keep up with this alarming situation, it has been suggested
`that genetic banks, mainly in the form of cell lines (preferably fi-
`broblast), should be established for critically threatened species.
`Scientists believe that those cells may be used to re-establish or
`expand the threatened population by somatic cell nuclear transfer
`(SCNT), a unique utilisation of preserved genetic material (Loi et
`al., 2008; Saragusty et al., 2016; Hildebrandt TB et al., 2018).
`SCNT has tremendous potential, not only for the rescue of
`endangered breeds and species (Loi et al., 2001), but also as a
`reproductive technology for genetically valuable farm animals or for
`generation of transgenic animals (Rodriguez-Osorio et al., 2009).
`Theoretically, SCNT is a simple technique, involving removal of
`nuclear DNA from an oocyte and its replacement with a somatic
`cell nucleus. Yet, despite its simplicity, the efficiency is basically
`the same since the first cloned animal was delivered (Wilmut
`et al., 1997). For this reason, many attempts to modify/improve
`SCNT efficiency have been reported (Iuso et al., 2013; Czernik et
`al., 2016). These improvements concerned the technical aspects
`(Wakayama et al., 2010, Czernik et al., 2016) as well as attempts
`as bulk or targeted modification of donor nucleus, before or af-
`
`ter embryo reconstruction (Wakayama, 2007; Wakayama and
`Wakayama, 2010; Iuso et al., 2015). This review will address the
`main improvements in the SCNT technique, as well as the role of
`mitochondria in cloned embryos and foetuses.
`Technical improvements in SCNT
`Improvement of the technique
`The traditional method of cloning (TDC) used in the majority
`of mammalian nuclear transfer laboratories involves the enucle-
`ation of matured (MII) oocytes, and subsequently replacing it (by
`different approaches) with a donor somatic nucleus (known as
`Nuclear Transfer) (Fig. 1A). The cytoplasm of human oocytes as
`well as laboratory animals like mouse and rat is clear and “trans-
`parent” under the microscope, which facilitates identification of
`MII chromosomes, hence their easy removal. On the other hand,
`the cytoplasm of large domestic animal’s oocyte is much darker,
`due to high lipid content, making it necessary to use fluorescence
`staining Hoechst 33342, ultraviolet (UV) exposure and cytocha-
`lasin B treatment during enucleation. Exposure to UV, however,
`has harmful effects on embryonic development (Gil et al., 2012).
`To avoid UV-related damages, Iuso and colleagues developed a
`new enucleation method in a large animal model, the sheep (Iuso
`
`Abbreviations used in this paper: IVF, in vitro fertilization; IVP, in vitro produced; Mfn2,
`mitofusin 2; MII, metaphase II; mtDNA, mitochondrial DNA; NR, nuclear repro-
`gramming; SCNT, somatic cell nuclear transfer; TSA, trichostatin A; UV, ultraviolet.
`
`*Address correspondence to: Pasqualino Loi. via R. Balzarini, 1, Campus Coste Sant’Agostino, 64100 Teramo, Italy. Tel: +39 0861 266856. E-mail: ploi@unite.it
` https://orcid.org/0000-0003-4631-7663
`
`#Current address: Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK.
`
`Submitted: 27 September, 2018; Accepted: 11 October, 2018.
`ISSN: Online 1696-3547, Print 0214-6282
`© 2019 UPV/EHU Press
`Printed in Spain
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`124 M. Czernik et al.
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`et al., 2013). Those authors showed significantly higher blastocyst
`rates when SCNT was performed without exposure of the oocytes
`to UV, as compared to the traditional method.
`Traditional method of nuclear transfer needs expensive equip-
`ment, as well as highly skilled personnel. To avoid that, several
`laboratories adopted a more economic procedure called Hand Made
`Cloning (HMC) (Vajta, 2007). The first successful HMC was done
`by Peura and colleagues (Peura et al., 1998). The technique then
`spread to the field of large domestic animal: buffalo (George et al.,
`2001), sheep (Zhang et al., 2013), horse (Lagutina et al., 2005),
`and pig (Du et al., 2007). In a further modification, researchers have
`successfully enucleated zona-free oocytes using sharp microblade,
`and attached the enucleated oocytes and donor cells using phy-
`tohemagglutinin, without any sophisticated equipment (Fig. 1B).
`Reconstruction of the enucleated oocytes can be performed
`by direct injection or by electrofusion of the somatic cell to the
`cytoplasm of the oocyte. There are two major methods for direct
`injection of donor cells’ nuclei. In the traditional way, membrane of
`the donor cell is lysed by repeated aspiration and ejection with a
`micro-capillary. The alternative way of reconstruction uses the Piezo
`device. The Piezo-driven technique, also known as the “Honolulu
`technique”, was first used by Wakayama, then at the University of
`Hawaii, to produce the first cloned mouse (Wakayama et al., 1998).
`The source of the donor nucleus is a crucial aspect when aiming
`to increase nuclear reprogramming efficiency. Most of the informa-
`tion available is on mouse model (Wakayama, 2007). The most
`commonly used donor cells, for ease of retrieval, are the cumulus
`cells (Wakayama et al., 1998). In addition to cumulus cells, tail tip
`fibroblasts (Wakayama and Yanagimachi, 1999), Sertoli cell (Ogura
`et al., 2000), foetal fibroblasts (Wakayama and Yanagimachi,
`2001a), embryonic stem cells (Wakayama et al., 1999), natural
`killer T cells (Inoue et al., 2005), and primordial germ cells (Miki
`et al., 2005) have all successfully been used. In large mammals,
`adult and foetal fibroblasts, cumulus cells, and embryo-derived
`cell lines were used (Kato and Tsunoda, 2010; Akagi et al., 2014).
`However, even though development to the blastocyst stage was
`sometimes reported to have been improved in nuclear transfer
`embryos, especially in those reconstructed with embryonic cells, the
`percentage of clones developing to term is still disappointedly low.
`Without deeply touching the issue, which was thoroughly dis-
`cussed in authoritative reviews, cloned embryos display a wide
`array of epigenetic alternation, including DNA methylation, histone
`acetylation, methylation, and non-coding RNA transcripts expres-
`sion (Matoba and Zhang, 2018). Correcting these shortcomings
`will certainly improve mammalian cloning efficiency. In the next
`section, we critically describe the solutions thus far adopted to
`minimise epigenetic alterations typically found in cloned embryos/
`conceptuses, and discuss the challenges ahead.
`Treatment with histone deacetylase inhibitors
`One of the biggest problems of cloned embryos is deacetylation
`of histones in the transferred cell nucleus. Acetylation of histones is
`generally associated with activation of gene transcription stemming
`from a more “open” chromatin structure. Zygotic gene activation, a
`crucial event at the beginning of zygotic genes transcription, occurs
`during early embryo development. Therefore, histone acetylation
`on transferred cell chromatin is important for proper zygotic gene
`transcription and embryo development that follows. However, it
`was reported that the level and state of several histone acetyla-
`
`tion marks in SCNT embryos chromatin were different from those
`in IVF embryos (Wang et al., 2007; Yamanaka et al., 2009). To
`improve cloning efficiency, several histone deacetylase inhibitors
`(HDACis) have been used (Fig. 1C). So far, one of the most ef-
`fective and commonly used inhibitors is Trichostatin A (TSA) that
`inhibits class I and II HDACs. Treatment of cloned embryos with
`TSA represses deacethylation on histones in embryo chromatin
`(Wang et al., 2007), improves the transcriptional activities at the
`2-cell stage, and increases the efficiency of full-term development
`in mice (Rybouchkin et al., 2006; Kishigami et al., 2006, 2007).
`TSA treatment of porcine cloned embryos also improved both pre-
`implantation and full development (Li et al., 2008).
`However, while the benefits arising from HDACis are incontest-
`able in mouse, its effects on large animal remains controversial.
`In fact, TSA treated bovine SCNT embryos showed improved pre-
`implantation development (Akagi et al., 2014), but no improvement
`in offspring rate (Sawai et al., 2012). Conversely, treatment of donor
`cells with TSA improved preimplantation development of embryos
`reconstructed with treated cells in buffalo (Luo et al., 2013). While
`in pigs, TSA treatment improves both pre-implantation develop-
`ment and offspring rate, in bovine and buffalo it improves only
`pre-implantation rate. Therefore, this method cannot be applied in
`large animal species.
`Impeding Xist expression
`One of the well-investigated epigenetic events in embryo devel-
`opment is X Chromosome Inactivation (XCI), in female mammalian
`cells. Since in mammals’ female cells have two X chromosomes,
`one copy is silenced so as to have the same proportion of gene
`products as that of males (dosage compensation). Xist, a non-
`coding RNA that is transcribed from the silenced X chromosome,
`is one of the better indicators when investigating the condition of
`XCI. In fertilized pre-implantation embryos, Xist shows monoallelic
`expression from the maternal X chromosomes; however, biallelic
`expression is observed in both inner cell mass and trophectoderm
`in cloned mice blastocysts (Nolen et al., 2005). Using Xist-deleted
`donor cells, to prevent this ectopic Xist expression, improved full-
`term development more than 7-8 times as compared to the normal
`procedure in mice (Matoba et al., 2011; Fig. 1D). Surprisingly,
`correction of Xist expression improves not only transcripts from
`X chromosome but also that from autosomes (Inoue et al., 2010).
`Moreover, injection of short interference RNA (RNAi) of Xist into
`reconstructed embryos, represses Xist expression transiently and
`improves full-term development of male (Matoba et al., 2011) but not
`female (Oikawa et al., 2013) cloned embryos. Ectopic expression
`of Xist is also observed in bovine (Inoue et al., 2010) and pig (Yuan
`et al., 2014, Zeng et al., 2016) cloned embryos and RNAi-mediated
`knockdown of Xist increases full-term development in male porcine
`cloned embryos (Zeng et al., 2016). Therefore, the partial conclu-
`sion is that Xist repression enhances cloned embryo development
`in males, and not only in the laboratory mouse.
`Histone lysine demethylase family member mRNA injection
`into embryos
`Methylation on histones is generally associated with repression
`of most of the genes transcription. Therefore, repressive marks on
`transferred donor cell chromatin, such as H3K9me3, are another
`epigenetic barrier in cloned embryos that prevents proper gene
`transcription after zygotic gene activation. Removal of histone
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`Progress in SCNT 125
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`A
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`B
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`C
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`D
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`E
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`F
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`Fig. 1. Improvements in somatic cell nuclear transfer (SCNT) techniques. (A) Traditional cloning (TDM). The MII plate is removed from the oocyte
`by micromanipulation, the enucleated oocyte is directly injected with the somatic donor nucleus and subsequently artificially activated. (B) Handmade
`cloning (HMC). The matured oocyte is exposed to Pronase to remove the zona pellucida; zona-free oocytes are enucleated by cutting with a micro sharp-
`blade, the somatic donor nucleus is aggregate with phytohemagglutinin and subsequently electro-fused with the ooplasm. Finally, the reconstructed
`embryo is artificially activated. (C) SCNT improvement by exposure of histone deacetylase inhibitors (HDACi) after reconstructed oocyte activation. (D)
`SCNT improvement by downregulation of Xist expression with siRNA. (E) SCNT improvement by ablation of the repressive histone mark, H3K9me3,
`through Kdm4d mRNA injection. (F) SCNT improvement by NT with protaminized somatic nucleus, through exogenous expression of human Protamine
`1 gene (pPrm1-RFP).
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`126 M. Czernik et al.
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`methylation has been achieved by injection of histone lysine de-
`methylase into reconstructed embryos (Matoba et al., 2014; Fig.
`1E). This treatment significantly improves full-term development
`of cloned mice (Matoba et al., 2014) and macaque monkeys (Liu
`et al., 2018) of both sexes. This application has even been used in
`human SCNT (Chung et al., 2015). It is expected that this method
`will be applied to other mammals in the near future.
`Treatment of reconstructed embryos with ascorbic acid
`It is reported that ascorbic acid treatment improves efficiency of
`reprogramming somatic cells to induced pluripotent cells in mouse
`and human (Esteban et al., 2010). This report prompted cloning
`scientists to test the possibility of improving nuclear reprogramming
`in SCNT embryos. Actually, vitamin C treatment of cloned embryos
`improves both preimplantation development and pregnancy rate
`in pigs (Huang et al., 2011). Moreover, combinations of the three
`components: deionized BSA, TSA and ascorbic acid treatment
`significantly improved cloning efficiency in mice (Miyamoto et al.,
`2017; Azuma et al., 2018). It is believed that the mechanism of
`this improvement is through repression of ROS and/or production
`and reduction of methylation on histone H3K9.
`Exogenous expression of the protamine 1 (Prm1) gene in
`somatic donor nuclei
`The male gamete is the perfect nuclear transfer device. Its DNA,
`tightly packed around protamines, confers the sperm nucleus a
`hydrodynamic shape to easily reach and fertilise the female gam-
`ete (Samans et al., 2014). Upon entering the oocyte, the sperm
`genome “springs out,” revealing its intrinsic totipotency. This may
`suggest that any successful NR strategy must mimic the nuclear
`reorganisation of the spermatozoon. In fact, this is the only nuclear
`formation the oocyte has evolved to deal with. Twenty years of
`experiments following the birth of “Dolly”, resulting in thousands
`of SCNT-derived embryos, showed invariably that the nuclear
`organisation of a somatic cells is rarely reset by the oocyte.
`Nuclear remodelling during spermatid maturation occurs
`through a time-regulated translation of mRNAs for histone variants
`that have accumulated earlier, in spermatogonia (Govine et al.,
`2013). Incorporation of such testis-specific histone variants into
`the chromatin leads to destabilisation of nucleosomes (Rathke
`et al., 2014). Subsequently, post-translation modifications of the
`histone variants further prepare the ground for the incorporation
`of transition proteins first, and then protamines, that compact the
`nucleus (Shabazianet et al., 2007). At present, it is impossible to
`repeat the stepwise spermatid nuclear remodeling in a somatic
`cell. Surprisingly, it has been recently shown that expression of
`protamine 1 alone is sufficient to compact the nucleus in a shape
`reminiscent of those of spermatids (Iuso et al., 2015; Czernik et
`al., 2016; Palazzese et al., 2018) (Fig. 1F). Furthermore, these
`authors observed that the protamine, when binding to the DNA,
`replaces the somatic histones, including H3K9me3, a critical
`epigenetic barrier of SCNT reprogramming (Iuso et al., 2015).
`Additionally, protaminised nuclei injected into enucleated, sheep
`oocytes resulted in an increase in blastocyst formation rate com-
`pared to traditional nuclear transfer (14% vs 4%, respectively).
`Those unique results, that demonstrate a radical reorganisation
`of somatic chromatin from histone to protamine, provide a prom-
`ising approach for improving mammalian cloning efficiency (Iuso
`et al., 2015).
`
`Embryo aggregation
`As we have described above, developmental outcomes of cloned
`embryos have improved by several kinds of treatments. However, the
`defects in the trophoblast cell linage, such as structural abnormali-
`ties and hyperplasia (Miki et al., 2009), have not been eliminated
`in any of these methods. Tetraploid embryo complementation is
`a widely-used application to prevent embryo lethality caused by
`placental dysfunction (Okada et al., 2007). Aggregation of inner
`cell mass (ICM) of diploid cloned embryos with trophectoderm (TE)
`derived from two or more tetraploid fertilised embryos can improve
`both full-term development ratio and placentamegaly in mice (Lin
`et al., 2011). Importantly, this improvement is not observed in case
`of whole embryo aggregation (Miki et al., 2009). Gestation period
`in large mammals is longer than in mice so this application might
`be more helpful for them.
`Mitochondria might affect the development of clones
`In this paragraph, we will address the limited success of the SCNT
`procedure from the mitochondrial perspective, an unexplored topic
`thus far. Investigations on mitochondria in SCNT procedures are
`limited to mtDNA hetero/homoplasmy in different tissues of cloned
`offspring (Lee et al., 2010). No data is available for an eventual
`role of mitochondrial dysfunction in developmental failure of the
`clones. Moreover, it is important to point that mitochondrial activity
`is strictly controlled by nuclear signals, suggesting that incomplete
`nuclear reprogramming in cloned nuclei might be responsible also
`for impaired mitochondrial function in cloned embryos/foetuses.
`Compared with nuclear reprogramming, which has been a lead-
`ing research topic over the last ten years, very few, if any, studies
`have focused on problems related to the association between
`mitochondria and nuclear reprogramming.
`In the next sub-sections, we will try to evaluate whether mito-
`chondrial dysfunction affect the embryo proper, the placenta, and
`foetal development.
`Mitochondria in SCNT embryos
`Following fertilisation and up to implantation, the embryo depends
`on the function of existing mitochondria, present in the oocyte at
`ovulation (Spikings et al., 2006). As cell division begins, the total
`number of mitochondria within each blastomere decreases due
`to dilution with no new mitochondrial biosynthesis (John et al.,
`2010). Early stage embryos do not express the nuclear-encoded
`replication factors required to multiply mtDNA. Mitochondrial DNA
`and mtDNA-nuclear DNA (nDNA) interactions might be responsible
`for the different phenotypes resulting from nuclear cloning. Lack
`of nuclear-mitochondrial interaction at the molecular lever can
`explain the potentially high development rate of early embryos
`[(mouse: 52,8% (Wakayama and Yanagimachi, 2001); sheep:
`27,3% (Wilmut et al., 1997); bovine: 69,4% (Wells et al., 1999)],
`even in distant inter-species nuclear transfer (mouflon: 30,4%
`(Loi et al., 2001). Replication defects of mitochondria may only be
`seen during embryogenesis, and play a role in post-implantation
`developmental defects. Consequently, any adverse influence on
`mitochondrial dysfunction (i.e., accumulation of mutational load to
`the mtDNA) may negatively impact the development of pre- and
`post-implantation cloned embryos.
`Mitochondria exhibit an interesting quality maintenance func-
`tion. They have numerous periods of fusion and fission. Active
`mitochondria are able to fuse with other mitochondria to transfer
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`components and maintain or improve the function of damaged or
`poorly performing members (Mouli et al., 2009).
`In mammals, three proteins are required for the fusion process.
`Two mitofusins are responsible for the fusion of the outer mem-
`brane, mitofusin 1 (MFN1) and mitofusin 2 (MFN2), while a single
`dynamic family member, OPA1, is required for inner membrane
`fusion. It is known that MFN2 plays a central role not only in the
`fusion process but is also responsible for key cellular functions
`such as oxidative metabolism, cell cycle maintenance, cell death,
`and mitochondrial axonal transport.
`The importance of mitochondrial fusion in early embryo de-
`velopment was reported by Mishra and Chan (2014). It was later
`demonstrated that MFN2 plays a crucial role in mitochondrial fu-
`sion, which is essential for mouse blastocyst formation (Jiang et al.,
`2015). Inappropriate mitochondrial activity at the pronuclear stage
`is associated with early developmental arrest (Blerkom et al., 2000)
`and such embryos show decreased expression of mitochondrial
`genes (Duran et al., 2011).
`Preliminary analysis of early mouse embryos stained with
`MitoTracker Green dye and evaluated by time-lapse microscopy
`showed minimal fusion process in SCNT blastocysts as compare
`to control groups (Czernik et al., unpublished data). Moreover,
`sheep and mouse early SCNT embryos show drastic differences
`in mitochondrial structure between SCNT and in vitro produced
`(IVP) blastocysts. Additionally, decreased density of mature mito-
`chondria, very high degree of cytoplasmic vacuolisation, numerous
`cytoplasmic vesicles and autophagosomes, as well as significantly
`lower expression of major mitochondrial, autophagic and apop-
`totic proteins were all observed in SCNT embryos (Czernik et al.,
`unpublished data).
`Mitochondria in SCNT placenta
`High frequency of first trimester losses, as well as late gesta-
`tion and post-natal losses, are observed with SCNT pregnancies
`as compared to in vitro fertilised and in vivo control pregnancies
`(Heyman et al., 2002). In most SCNT pregnancies, high rate of
`foetal/embryonic loss is associated with placental malformation
`(Loi et al., 2006). Placental insufficiency resulting from abnormal
`cotyledon formation, decreased numbers of cotyledons, placental
`degeneration and reduced placental vascularisation are considered
`to be the cause for diseases typically documented in SCNT foetuses
`and neonates, including respiratory distress, malnutrition, and car-
`diopulmonary disease (Loi et al., 2006). The placenta has a crucial
`role in maternal control of foetal development and it represents
`an interface between the maternal environment and the foetus.
`Furthermore, many of the genes that regulate placental develop-
`ment also regulate foetal brain development (Murphy et al., 2006).
`As was mentioned before, fusion of mitochondria plays crucial
`role in their proper function. It has been shown that mice, defi-
`cient in MFN2 protein, die in utero at mid-gestation of placental
`deficiency due to placental abnormalities, particularly disruption of
`the trophoblast giant cell layer (Chen et al., 2003). Additionally, it
`has been shown that low expression of MFN2 in human placentas
`is associated with mitochondrial damage in placental cells and
`unexplained miscarriage (Pang et al., 2013). Recently, Czernik et
`al., (2017) reported that deregulated expression of mitochondrial
`proteins (MFN2 and BCNL3L) cause abnormalities in early preg-
`nancy placenta in sheep. Abnormalities were mainly presented as
`damaged and malformed mitochondria, as well as swollen endo-
`
`plasmic reticula (Czernik et al., 2017). Similar findings were also
`reported by Wakisaka and colleagues in mouse cloned placentas
`(Wakisaka et al., 2008). These observations clearly suggest that
`mitochondria in SCNT placentas do not work properly and this
`may negatively affect SCNT embryo development.
`Mitochondria in SCNT foetuses
`Many SCNT embryos are lost due to gestation and neonatal
`failures. Birth defects and high post-natal losses are seen in cloned
`cattle, sheep and pigs as well as in laboratory animals. Oversized
`livestock at birth (Bertolini et al., 2002), cloned calf syndrome
`(Wells et al., 2004) or more usually the large offspring syndrome
`(Young et al., 1998) are frequently observed in sheep and cow.
`Young and co-workers reported that oversized livestock at birth is
`related to imprinting dysregulation of IGF2R (Young et al., 2001)
`but full explanation of the underlying causes is still missing. Given
`the finding that mitochondria play a role in obesity (Ritov et al.,
`2005), it may well be that mitochondria abnormalities are involved
`in oversized live-stock neonates associated with cloned animals.
`Major health problems include respiratory distress, circulatory
`problems, immune dysfunctions and kidney and heart failure. More-
`over, problems with movement and balance have been observed
`in cloned animals, displaying uncoordinated limb movements,
`and therefore movement primarily by writhing on their abdomens
`(Wells et al., 2004).
`In mouse mutants, homozygosity for both Mfn2 or Mfn1 is lethal at
`early stages of development (Chen et al., 2007). Cerebellum-specific
`inactivation of Mfn1 or Mfn2 has been achieved by crossing Mfn1
`loxP or Mfn2 loxP mice. These mice express a cerebellum-specific Cre
`recombinase-driven promoter (pMeox2-cre). Mfn1 inactivation in
`the cerebellum resulted in mice with normal growth, development,
`and fertility. However, cerebellum-specific inactivation of the Mfn2
`gene resulted in one-third of the mice dying within one day after
`birth and the surviving animals showing severe defects in move-
`ment and balance. The cerebellum of the Mfn2-deficient mice was
`only 25% the size of control mice at post-natal days 15-17; this
`disparity was associated with reduced and deteriorating Purkinje
`cells and increased apoptosis of granule cells (Chen et al., 2007).
`The mouse Mfn2 pMeox2-cre mutants-associate abnormalities
`overlap with those observed in neonatal clones.
`Mitochondria and nuclear reprogramming
`The mitochondrial genome transcribes only 13 proteins, while the
`remainder mitochondrial proteins (1500-2000) are encoded by the
`nucleus, where the crucial mitochondrial genes have been trans-
`ferred to benefit from more accurate (error-free) DNA replication.
`Critical nuclear and cytoplasmic interactions may be determined by
`the mitochondria. There is a great deal of communication between
`the nuclear and mitochondrial genomes, and this communication
`strictly controls mitochondrial function (Chappel, 2013). It is im-
`portant to mention, once again, that the major reason for the low
`efficiency and abnormalities observed in SCNT embryos/foetuses
`is incomplete somatic cell Nuclear Reprogramming (NR). It might
`be that mitochondrial genome is not activated properly due to in-
`correct NR and this causes malfunction of the mitochondria, and
`hence abnormalities in cloned placentas and foetuses.
`These findings suggest that mitochondria dysfunction might oc-
`cur following nuclear transfer due to failure in nucleus-cytoplasmic
`interaction leading to failed nuclear remodelling. Proper nuclear
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`128 M. Czernik et al.
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`remodelling is required to initiate mitochondria differentiation. Work
`report negative consequences of nuclear-cytoplasmic interaction
`for foetal development after bovine nuclear transfer, indicating com-
`plex oocytes cytoplasm-dependent epigenetic modifications and/
`or nuclear DNA-mitochondrial disrupted interactions (Heindleder
`et al., 2005). It is also possible that mitochondrial dysfunction
`may contribute to activation of the apoptosis cascade, resulting in
`developmental defects and abnormalities (Schatten et al., 2005).
`To understand mitochondrial-nuclear interactions in reconstructed
`embryos more detailed studies will be needed.
`Conclusions
`Current efficiency of SCNT hampers its practical application. The
`strategies put forth to improve its efficiency have had a negligible
`effect in farm animals, and minimal advancements have been
`achieved only in the mouse. Moreover, the unexpected mitochondrial
`dysfunctions in cloned embryos, reported for the first time by our
`group (Czernik et al., 2017), adds a further level of complication
`to cloning research. Clearly, it appears unlikely that all the biologi-
`cal constraints impairing cloning could be removed by a single
`treatment/protocol. Genome wide nuclear remodelling remains a
`priority in our opinion. Ideally, the perfect nuclear reprogramming
`strategy should work across all species, not only mammals. It is
`worthy to mention here that 24 species, including amphibian, fish,
`mammals, and insects have been successfully cloned so far. The
`message that this review would like to convey is that strategies
`described above have a potential to make a difference in nuclear
`reprogramming efficiency. The most promising strategies, in our
`opinion, are those acting on the entire genome, such as the forced
`expression of histone demethylases, or conversion of the chromatin
`structure typical for somatic cells to the spermatid-like structure
`(Fig. 1). Then, other issues, like mitochondrial dysfunction in normal
`clones or in interspecies SCNT, or lack of activation of the zygotic
`genome in the latter case, will need to be pinned down.
`SCNT remains, after all, the most powerful way to reset the
`epigenetic memory in somatic cells. Any advancement in clon-
`ing research will have unquestionable benefits for regenerative
`medicine, species conservation, multiplication of desired geno-
`types or phenotypes, and lastly, for the introduction of the latest
`genomic research advancements like genome editing into farm
`animal breeding.
`
`Acknowledgments
`This project has received funding from the European Union’s Horizon
`2020 Research and Innovation Programme under the Marie Skłodowska-
`Curie grant agreement No. 734434, from National Science Centre, Poland
`by the grant No. 2016/21/D/NZ3/02610. The authors participate in the COST
`Action CA16119. The authors dedicate this paper to Andrzej K. Tarkowski,
`unforgettable titanic scientist in Developmental Biology.
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