Human Reproduction Update 1998, Vol. 4, No. 6 pp. 791–811
European Society of Human Reproduction and Embryology
How identical would cloned children be? An
understanding essential to the ethical debate
R.G.Edwards1 and Helen K.Beard
Human Reproduction Journals, Moor Barns Farm, Madingley Road, Coton, Cambridge CB3 7PG, UK
TABLE OF CONTENTS
Key words: cloning/epigenesis/ethics/human/
monozygotic twins
ality, cloning was bracketed with other non-permitted activities including the creation of animal/human hybrids,
genetic modifications of embryos and, strange to say, the
cryostorage of embryos. Alarm was sustained by films such
as ‘Boys from Brazil’.
Underlying these concerns is the fundamental assumption
that cloned children will be identical to their nuclear donor or
their nuclear siblings. In this commentary, we consider the
scientific basis for this assumption. Clones, defined as ‘a
group of living organisms sharing the same nuclear gene set’
(opinion of the group of advisors on the Ethical Implications
of Biotechnology to the European Commission, unpublished) are essentially monozygotic twins formed by
biological manipulation. Two experimental methods have
been used in animals to produce cloned embryos: embryo
splitting and nuclear transfer. While embryo splitting gives
rise to monozygotic twins at a high efficiency, up to 30% in
cattle (Williams et al., 1984) and sheep (Chesne et al., 1987),
and may be considered as an acceptable application to
humans in the next decade, we confine our discussion to
cloning by nuclear transfer since it is this process which has
generated such adverse reactions. We outline details of the
cloning process, and explore the effects of uterine and
environmental factors on biological variability in cloned
animals and monozygotic human twins.
Introduction
Principles of cloning embryos
The prospect of cloned children, especially in large identical
groups, raises widespread alarm. In virtually every country
that has passed legislation on assisted human conception, a
clause has been inserted to ban the application of cloning to
humans on pain of severe penalties. Concern about cloning
arose after the initial studies in amphibians, and re-emerged
when human eggs were first fertilized in vitro. There were
abounding fears about self-cloning, identical football teams,
and fetuses cloned to produce spare organs for the nuclear
donor. Considered an affront to human dignity and individu-
Studies on nuclear cloning in mammals followed initial
work in amphibians by Briggs and King (1952) and Gurdon
(1964). The method involves the fusion of a nuclear
karyoplast taken from cells in various tissues with an
enucleated metaphase II oocyte or an enucleated pronucleate
egg (Baranska and Koprowski, 1970; Lin et al., 1973;
Bromhall, 1975; Tarkowski and Balakier, 1980; Illemensee
and Hoppe, 1981; McGrath and Solter, 1986; Willadsen,
1986; Tsunoda, 1987; Marx, 1988; Szollosi et al., 1988).
Metaphase oocytes are enucleated by excising their spindles
Introduction
Principles of cloning embryos
Cytogenetic modifications and abnormal
development after nuclear fusion
Epigenetic regulators in cloning
Practical aspects of cloning
Monozygotic human twins: nature’s clones
How closely would cloned children
resemble their parents and each other?
Conclusions
Acknowledgements
References
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The ban on human cloning in many countries worldwide is founded on an assumption that cloned children
will be identical to each other and to their nuclear
donor. This paper explores the scientific basis for this
assumption, considering both the principles and practice of cloning in animals and comparing genetic and
epigenetic variation in potential human clones with
that in monozygotic twins.
1To
whom correspondence should be addressed
792
R.G.Edwards and H.K.Beard
and chromosomes, while pronuclei are excised from fertilized eggs. DNA dyes applied to excised ooplasm are used to
verify the full removal of all chromosomal material (Bondioli, 1993). Up to one-quarter of the ooplasm can be excised
during this process in sheep (Smith and Wilmut, 1989). The
donor karyoplast, containing the nucleus with some cytoplasm, is then placed in the perivitelline space, adjacent to
the oocyte membrane, and fusion and activation are induced
by electrical, viral or chemical stimulation (Stice and Robl,
1988; Cheong et al., 1993; Prather, 1996). Cytochalasin B
and other microtubule blockers are necessary to control cytoskeletal movements and allow enucleation (Stice and Robl,
1988; Smith and Wilmut, 1989; Cheong et al., 1993; Kwon
and Kono, 1996; Prather, 1996).
Ooplasmic factors reprogramme the nucleus, conferring
on it an ability to regulate embryonic growth to full term.
Understanding of the key processes is still incomplete, but
underlying mechanisms are being clarified. Blastomere karyoplasts are widely used, and others are taken from fetuses
or adults. Nuclei in 2- and 4-cell embryos are totipotent,
capable of supporting full-term development and this characteristic may require the expression of particular genes
including non-specific alkaline phosphatase, Tnap, transcription factors, Oct3/4, proto-oncogene, c-kit, and the
DNA methyltransferase, Mt (Urven et al., 1993). In most
mammals, blastomeres become committed by the
8–16-cell stage or even earlier so that later stages are no
longer totipotent, and their nuclei may have to reprogrammed during cloning.
Status of the recipient oocyte
The status of the recipient oocyte is an important factor in
cloning. Ooplasm changes its properties during maturation
and fertilization as the cell is released from metaphase II
arrest and activated at fertilization by a series of calcium
discharges (Swann and Ozil, 1994) possibly involving
cortical rotation (Edwards and Beard, 1997). Maternal
RNA transcripts then control development in the first few
cleavage divisions until zygotic activation (Braude et al.,
1979, 1988). These transcripts must direct the cloned nucleus as it assumes control of early embryonic phases. Spatial considerations, e.g. polarization of ooplasm and
localized maternal mRNAs, could be essential for normal
growth (Gardner, 1996, 1997; Antczak and Van Blerkom,
1997; Edwards and Beard, 1997), but this aspect has not
been considered in relation to cloning.
The existence of cytostatic factor (CSF) and maturation
promoting factor (MPF) in oocytes have been known for
two decades from work in amphibians (Masui and Markert,
1971). The components of these factors are now better
understood and appear to be ubiquitous, at least in vertebrates. MPF contains a complex of cyclin B and the protein
kinase p34cdc2 (Gautier et al., 1988) and CSF activity is
decided by the activity of the mos protein, pp39mos, the
product of the c-mos proto-oncogene (Sagata et al., 1989).
These two factors interact. MPF is activated by phosphorylation of cdc2 kinase (Gautier et al., 1991) and inactivated by degradation of cyclin B. Mos protein stabilizes
MPF by preventing cyclin B breakdown, possibly indirectly via interaction with the mitogen-activated protein kinase
(MAPK) pathway (Sagata, 1997). The role of mos protein,
expressed so specifically in maturing oocytes, is thought to
be prevention of DNA replication prior to fertilization (Sagata, 1996, 1997). At fertilization, and probably after electrostimulation, the calcium dependant neutral protease,
calpain is activated (Watanabe et al., 1989), degrading mos
protein, and destabilizing the MPF complex (Edwards and
Brody, 1995).
Histone H1 kinase activity has been used in reconstructed bovine embryos to measure MPF concentrations
which decline rapidly after activation, e.g. down to 30%
after 1 h and 20% after 2 h (Campbell et al., 1993) although
reactivation may occur after a single activation pulse (Collas et al., 1993). Since MPF is associated with the spindlechromosome complex (Fulka et al., 1996; Sagata, 1996) it
may be depleted by the process of enucleation prior to
activation. Several authors stress the importance of MPF in
nuclear envelope breakdown, premature chromosome condensation (PCC) and reorganization of the cytoskeleton
(Campbell et al., 1993; Prather, 1996), while Leno et al.
(1992) suggest that nuclear membrane breakdown may
occur with G1 donor nuclei but not with those in G2. Dissolution of the donor nuclear envelope presumably proceeds in a manner resembling disassembly of the sperm
membrane during normal fertilization (Poccia and Collas,
1997). MPF may also play a beneficial role in remodelling
G2-phase donor nuclei (Fulka et al., 1996).
Thus cloning may best be achieved with aged MPF-deficient oocytes rather than fresh oocytes, and with enucleated
fertilized or activated eggs rather than with enucleate metaphase II oocytes since MPF decays during such a delay
(Prather et al., 1987; Campbell et al., 1993, 1994; Prather,
1996). Some investigators prefer to clone activated oocytes
some hours later, e.g. during their S phase, when MPF
concentrations have subsided (Campbell et al., 1993;
Kwon and Kono, 1996). In bovine embryos, the combination of ageing, cooling and enucleation results in low MPF
activity but high MAP kinase activity (Gall et al., 1996)
Matching cell cycles in donor nuclei and recipient
enucleated oocytes may be advantageous, particularly
How identical would cloned children be?
when MPF concentrations are high, to yield good rates of
growth to full term (Gurdon, 1964; Chastant et al., 1996).
Transferred nuclei must interact with a sequence of
cytoplasmic activators and repressors which control
normal zygotic gene transcription (Majumder et al., 1993;
Yang et al., 1995; Davis et al., 1996). Metabolic variations
such as methylation of insulin-like growth factor (IGF) II
receptor could occur in cloning as in parthenogenetic
mouse embryos stimulated by electroactivation (Kono
et al., 1996). The whole procedure must involve stress, and
possibly the production of heatshock proteins (Kurtz et al.,
1986) in far greater amounts than under normal conditions
of culture (Gardner and Leese, 1990; Conaghan et al.,
1993; Vernet et al., 1993; Christians et al., 1995). Stress
invokes numerous effects, such as changes in protein
synthesis in embryos growing in vitro (Reik et al., 1993) as
well as apparent long-lasting effects such as large
birthweights in cattle and sheep (Walker et al., 1992;
Behboodi et al., 1995). Such large birthweights are more
common after cloning (Keefer et al., 1994; Renard, 1998).
Cloning of isolated plant stem cells also involve stress
responses to growth factor starvation, these responses
involving somatic meioses, homeotic transformation and
somatic embryogenesis in response to this form of
starvation (Nuti Ronchi, 1995). Isolated plant protoplasts
display somatic meiosis when fused after removal of their
chitinous cell membranes, and have been used to establish
wide and variable crosses between species (Lowe et al.,
1996; Parkonny et al., 1997). All these observations
illustrate the profound effect of environmental factors on
early development and the importance of culture
techniques and conditions on the outcome of cloning.
Status of the donor karyoplast
Initial studies on cloning in mammals utilized nuclei from
blastomeres of preimplantation embryos, and shifted later
to nuclei from fetal, and adult tissues. The blastomeres are
in their formative periods of cellular differentiation and sex
determination (Antczak and Van Blerkom, 1997; Edwards
and Beard, 1997), yet they have the advantage of
containing nuclei capable of reverting easily to an
oocyte-like condition. Adult nuclei presumably need to
reverse changes imposed by ≥30 cell cycles, i.e. the
number of cycles estimated to occur in the transition from
single cell to a 1 × 109 cell adult, so it is hardly surprising
that cloning is more successful with younger nuclei.
Two-cell nuclei are more effective than 8-cell nuclei in
mice (Cheong et al., 1993), nuclei from rabbit inner cell
mass give better development than granulosa cell nuclei
(Collas and Barnes, 1994), and embryonic rather than adult
793
cell nuclei give higher success rates in sheep and cattle
(Keefer et al., 1994; Wilmut et al., 1997). The birth of
Dolly (Wilmut et al., 1997), whose cloned status is now
fully confirmed by DNA microsatellite analysis (Ashworth
et al., 1998) and DNA fingerprinting (Singer et al., 1998)
and supported by recent work in mice (Wakayama et al.,
1998) decisively widens the scope of cloning, to the use of
fully differentiated adult nuclei.
Successive nuclear modifications during growth to
adulthood must presumably be reversed in cloning. These
include DNA methylation and imprinting (Monk et al.,
1987), X inactivation, histone acetylation (Turner, 1991),
and modifications in other nuclear proteins (Pinto-Correia
et al., 1995), enzyme activation (Worrad et al., 1995), and
telomere shortening (Harley et al., 1990). Telomeres,
centromeres and chromosome-specific subsatellite regions
display their own typical arrangements in interphase
somatic nuclei, e.g. centromeric DNA can be held on the
nuclear envelope, dispersed throughout the nucleus or
concentrated at one pole particularly in S and G2 phases,
although there are wide species differences (Vourc’h et al.,
1993). These nuclear modifications may have to be
reversed in cloning. Genetic modifications arising in adult
nuclei during growth could include somatic mutagenesis
(presumably at a rate of 1 in 109 base pairs per nucleus),
mitotic errors, deletions and disomy. These factors will be
present in cloned nuclei and will influence embryonic
growth. Reversal of these modifications, together with
epigenetic effects of cloning, could cause wide differences
from the original genotype of the nuclear donor.
The process of nuclear de-differentiation and
remodelling is not understood although several nuclear
markers have been studied in order to identify changes
occurring in transferred nuclei. Nuclear lamins A and C,
polymerize during early cleavage stages in mice, pigs and
cattle, and during nuclear swelling in cloned embryos
(Prather et al., 1989; Kubiak et al., 1991). The appearance
of phosphorylated polypeptides perhaps involved in DNA
repair, replication, recombination and transcription, is
another indicator. They normally appear 2 h after
fertilization in rabbit eggs during sperm head decondensation, and are first formed in clones at 10 h
post-fusion (Pinto-Correia et al., 1995). Blastocoel
formation can be delayed in rabbit clones (Stice and Robl,
1988). Nucleolar reprogramming is delayed in some
cloned 32-cell rabbit embryos, since fibrillarin forms in
only a proportion of cloned blastomeres, in comparison
with all cells of normal 32-cell embryos. Fibrillarin
associates with nucleolar small ribonuclear RNA and
marks the onset of nucleolar transcription (Pinto-Correia
et al., 1995), and its delayed synthesis in clones may be the
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R.G.Edwards and H.K.Beard
consequence of defects arising in nucleolus organizer
chromatin, which is heterochromatic. Changes in nucleolar
ultrastructure and a large increase in free ribosomes
suggested that reconstituted rabbit embryos at the 32-cell
stage had disrupted ribosomal transport mechanisms
(Kanka et al., 1996). In contrast, some cell surface antigens
are expressed simultaneously in fertilized 32-cell embryos
and clones (Van Stekelenburg-Hamers et al., 1994).
The de-differentiation of somatic cells in vitro may help to
understand cloning, even though it is induced via the cell
surface rather than by cytoplasmic messengers. Both
systems involve gross changes in gene expression.
Modifications to tissue culture substrates has been shown to
alter phenotypic expression in a variety of systems. Chick
retinal pigmented epithelial cells (PECs) de-differentiate,
proliferate and re-differentiate into lens cells as they activate
α-, β- and γ-crystallin genes when placed in media
containing phenylthiourea and hyaluronidase, whereas
collagen substrates inhibit this transition. The expression of
certain PEC-specific genes such as MMP115 and pP344 is
repressed as the cells de-differentiate and re-activated as they
revert to PECs (Agata et al., 1993; Mazaki et al., 1996).
Culture media containing glucocorticoids help to up-regulate
expression of the breast cancer genes, BRCA1 and BRCA2 in
mammary
epithelium
during
proliferation
and
differentiation, but not when deprived of serum (Rajan et al.,
1996).
Wide morphological changes can be induced in vitro.
When primary explants of bovine mammary epithelial
cells are cultured on fibronectin or collagen, rather than
laminin, an epithelial pavement can form over 4 weeks,
with tight junctions, desmosomes, apical villi and a high
transepithelial electric resistance (Delabarre, 1997). When
placed on plastic or connective tissue (Matrigel) substrates,
fetal enterocytes de-differentiate, whereas they remain
fully differentiated, displaying typical enzymes in their
brush border membranes, if cultured on laminin or
co-cultured with fibroblasts (Sanderson et al., 1996).
Cartilage cells de-differentiate when cultured to high cell
densities with bone morphogenic protein on various
matrices and can be switched into specific cell types for use
in reconstructive surgery. Human cells are being placed on
biodegradable mesh scaffolds with specific substrates in
order to construct replaceable organs such as skin, breast,
nipples, ears and cornea in work approaching clinical trials
(Vacanti, 1988; Sittinger, 1995; Cao, et al., 1997). With
both nuclear de-differentiation and cloning, further
experimentation is required to understand the signals and
responses involved in these processes.
Synchronization of donor and recipient cell cycles
Most forms of cloning have utilized enucleated metaphase
II oocytes. Results with cloning blastomere nuclei vary
according to the cell cycle stage in the donor and recipient.
Some investigators use donor nuclei in G1/S (Cheong
et al., 1993; Pinto-Correia et al., 1995); others prefer G0
nuclei from confluent cell cultures in ‘starvation culture
media’ (Wilmut et al., 1997). Metaphase nuclei are used
rarely but produce normal embryos and offspring in mice
(Kwon and Kono, 1996). Nuclei from mouse 2–8-cell
embryos in G1, S and G2 phase produce blastocysts at a rate
of 78, 0 and 21% respectively, and G1 nuclei yield 18–29%
young per transferred embryo (Cheong et al., 1993).
Reservations are expressed about these results since the G1
phase is very short or absent in blastomere nuclei
(Pinto-Correia et al., 1995; Kwon and Kono, 1996), and it
is notable that Kwon and Kono (1996) could not repeat the
work of Cheong et al. (1993). Cloning G0 nuclei was
successful with Dolly following a G0 induced by cell
starvation in culture. Usually, G0 occurs during or at the
end of G1, and the activation of the nucleus during cloning
would resemble the natural progression from G1 to early S
phase of the mitotic cycle or gamete fusion to post-S phase
in a fertilization cycle (Figure 1a,b). G0 can also arise at the
S–G2 transition in some cell types, e.g. Drosophila nurse
cells (Su et al., 1988). It is not clear which of these was the
G0 phase used by Wilmut et al. (1997).
Synchronization of the cell cycle phases in the donor
nucleus and recipient oocyte may be very important for the
success of cloning. When MPF concentrations are high in
fresh enucleated oocytes, PCC and nuclear envelope
breakdown occur allowing reinitiation of DNA synthesis
(Barnes et al., 1993; Campbell et al., 1993). When donor
nuclei are in G2 phase, DNA replication must be completed
(centromeric DNA synthesis) prior to PCC. Aneuploidy
will result from complete re-replication of DNA in a single
nucleus, although the 4S status and normal cleavage will
follow extrusion of a polar body (Figure 1c).
PCC was first described in HeLa cells fused at different
stages of the mitotic cell cycle (Johnson and Rao, 1970).
The fate of chromosomes following PCC depends on the
cell cycle stage of the karyoplast. Early S nuclei can form
rare double spindles, sticky chromosomes, micronuclei
and chromosomal mosaicism. With S phase nuclei,
chromosome pulverization may occur as was observed in
the original studies on HeLa cells (Johnson and Rao, 1970).
Late S nuclei can produce irregular metaphase plates, and
half or disorganized spindles, chromatin patches and
How identical would cloned children be?
795
Figure 1. Models of the normal mitotic cell cycle, events at fertilization and cloning in relation to DNA content and chromosome number, as
well as chromatid and centromere structure. S values indicate DNA levels which double in the S phase as chromatids replicate. Maternal (M) and
paternal (P) chromatids are indicated. (a) DNA content during the normal mitotic cell cycle. The extended period of DNA synthesis in heterochromatic, including centromeric regions, is indicated. Cell division at metaphase restores 2S in daughter cells. (b) Modifications of the cell
cycle and DNA content during fertilization. Metaphase II oocytes and first polar bodies are 2S. At fertilization, the oocyte and second polar body
become 1S, and the spermatozoon introduces 1S. Fertilized eggs thus become 2S immediately after fertilization in their G1 phase, and 4S after
their S phase. The first cleavage division produces 2S in each daughter blastomere. (c) Cloning nuclei in late S and G2 phase. Successive illustrations show the consequences of fusing donor karyoplasts with recipient enucleated oocytes in meiosis metaphase II. A single chromosome pair is
shown. Nuclei in late S are approaching 4S and are replicating chromatids, while those in G2 have completed this process. Neither stage has
replicated centromeres since heterochromatic regions replicate late in the cell cycle. Nuclei presumably undergo premature chromosome condensation (PCC) and replicate centromeres before the onset of spindle formation. A complete DNA synthesis in the pronucleus involving chromatids and centromeres would establish 8S. If a polar body and a pronucleus are formed, they will each be 4S and the eggs would be ready for
normal cleavage. If polar body expulsion is prevented, the egg forms one or two pronuclei, with a total of 8S, and this could be incompatible with
normal development. Species differences appear to result in polar bodies being expelled in cloned mouse eggs but not in those of farm animals
and rabbits.
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R.G.Edwards and H.K.Beard
larger-sized chromosomes. Nevertheless, the overall
impression is of similar events in all of these stages, so that
many cloned embryos inherit normal chromosome counts
and morphology and cleave normally (Collas et al.,
1992a,b).
The situation in cloning is entirely different if donor
nuclei are fused several hours after oocyte enucleation and
activation, or with enucleated pronucleate eggs. Many
recipient eggs will then be in S, a perfect match for donor
nuclei also in their S phase. Campbell et al. (1993, 1994)
stress that PCC does not occur at all under these conditions,
and that oocytes activated some hours before fusion are
‘universal cytoplast recipients’. Almost all of their nuclei
were in S, and therefore synchronous with the recipient
egg. Fusion several hours after activation is also effective
in cattle, and produces few chromosomal anomalies in the
embryos (Barnes et al., 1993). Mouse pronuclei in S phase
have been recloned into recipient enucleated pronucleated
eggs which were also in or close to S phase, and have
yielded very high rates of nomal growth among the
recloned offspring (Figure 2) (Kwon and Kono, 1996).
Cell cycle problems could still arise with G2 and M donor
nuclei fused with enucleated recipient eggs in S phase.
They could be forced back into an abnormal S phase
involving centromeric regions or even whole
chromosomes. Wilmut et al. (1997) evidently abandoned
the universal recipient system when cloning Dolly, and
instead returned to enucleated metaphase II oocytes and G0
donor nuclei.
Many unknowns about cloning remain to be solved. Cell
cycle phases stated by many investigators may not be
formally correct, since G1 is brief or non-existent in
blastomere nuclei. G1 phase must be induced artificially by
arresting blastomeres at metaphase, e.g. using nocodazole.
The embryos which are to produce the donor nuclei are
then transferred to medium without nocodazole and
containing hydroxyurea, and checked hourly for cleavage;
immediately after cleavage, the temperature is lowered on
the assumption that blastomeres now in G1 or at the G1/S
boundary will remain arrested there (Campbell et al.,
1993). Whether this procedure produces the desired result
is debatable, since the S phase can begin before mitosis is
completed in some types of cell, or nuclei may enter the S
phase during the delay in extracting and fusing them.
Cytogenetic modifications and abnormal
development after nuclear fusion
Nuclear and chromosomal situations
Major developmental, chromosomal and centromeric
epigenetic modifications arise in cloning, particularly
Figure 2. Recloning pronuclei into enuleated pronucleate eggs in
mice. The method is highly successful since the donor nucleus and
recipient cytoplasm are completely co-ordinated (Kwon and Kono,
1996). There are no reports on the occurrence of premature chromosome condensation (PCC) with this form of cloning. Recipient ooplasm does not apparently impose meiotic-like phenomena on the 4S
donor chromosomes, and centromere synthesis is apparently unnecessary. Completion of the mitotic cycle will establish 2S in the egg
and in the polar body. The egg will become 4S during pronuclear
DNA synthesis and, therefore, establish correct conditions for cleavage. PVS = perivitelline space.
when recipient oocytes and donor nuclei differ in their cell
cycle stages. Abnormalities, such as polyploidy (Campbell
et al., 1993) and disomies as well as imprinting defects
may arise and this could account for some of the high levels
of embryonic and fetal loss.
The production of either two ‘pronuclei’, a pronucleus
and one or two polar bodies, or a single (diploid?)
pronucleus after nuclear transfer could be influenced by the
use of nocodazole or cytochalasin which impair tubulin
function and polar body extrusion (Kwon and Kono,
1996). Species variations seemingly arise, since polar
bodies are apparently not regular features of cloning in
rabbits (Stice and Robl, 1988; Collas et al., 1992), sheep
(Robl et al., 1987; Collas et al., 1992) and cattle (Prather
How identical would cloned children be?
797
Centromeric DNA synthesis
Figure 3. Segregation of chromatids in a chromosome pair as premature chromosome condensation (PCC) and metaphase arise during
cloning. Maternal (M) and paternal (P) chromosomes are shown individually, with their chromatids.
et al., 1987; Bondioli et al., 1990; Campbell et al., 1993,
1994). Polar body extrusion apparently leaves some eggs
with insufficient chromosomes for a normal first cleavage
division (Cheong et al., 1993). The observation that
cloned embryos extrude polar bodies, is astonishing and
perhaps a further sign that oocyte microtubules control
donor chromosomes and impose a late form of meiosis.
These successive changes presumably utilize ooplasmic
factors and chromosome-associated centromeres,
centrioles, and lamins. Normally, the sperm centriole is
involved in spindle formation in the embryo, and it seems
that the cloned nucleus can assume this function. Nucleoli
may have to adapt to a different function during cloning,
initially being inactivated and then producing compounds
such as nucleolin and fibrillarin (Baran et al., 1995).
Chromosomal damage, as yet undefined, may arise
during PCC (Collas et al., 1992; Campbell et al., 1994;
Kwono et al., 1996; Prather, 1996). Chromosome
association or pairing, the formation of 0, 1 or 2 polar
bodies (Kwon and Kono, 1996), unipolar and uneven sized
spindles (Collas et al., 1992) and other disorders may
distort normal chromatid separation to the poles after PCC.
Meiotic-like phenomena similar to PCC, but not
chiasmata, occur in plant cells cloned from individual stem
cells (Nuti Ronchi, 1995). Anomalous segregation could
cause disomy and other wide genetic variations in the
cloned nuclei (Figure 3). Disomy, which is of immense
clinical significance for various cancers, involves two
simultaneous events: loss of one parental chromosome set,
and a (simultaneous?) doubling of the other. Perhaps
unusual forms of centromere synthesis in somatic cells, just
as in PCC, are responsible for these complex chromosomal
events.
A poorly understood event of centromeric DNA synthesis
has been observed in cloned rabbit embryos just before
PCC (Pinto-Correia et al., 1995). Two distinct phases of
DNA synthesis thus occur in cloned metaphase II
recipients. One phase occurs before or during PCC when
oocytes are ‘still at transient spindle stage’ and is restricted
to centromeric regions. The second phase occurs after PCC
in pronuclei and involves a complete DNA synthesis
including previously-replicated centromeric regions.
Pinto-Correia et al. (1995) suggest that this could lead to
developmental defects, although many of their embryos
developed normally to blastocysts. Although steps are
often taken to synchronize donor nuclei in the early S
phase, the DNA synthesis that began in cells of the original
donor embryo was probably being completed just after
fusion occurred, since centromeric and other
heterochromatic regions are known to prolong DNA
synthesis until the M phase (Wolfe, 1981). This situation
also explains the low-level synthesis of DNA until PCC
(reported by Collas et al., 1992a) and which was possibly
due to the synthesis of heterochromatic centromeric DNA
late in the cell cycle of the donor nucleus. Other forms of
heterochromatin could also replicate very late in the cell
cycle. With blastocyst nuclei, for example, one X
chromosome is inactivated in female embryos by actions of
the gene Xist, and this heterochromatic chromosome
contains less acetylated isoforms of histone H4 and
replicates late in the S phase (Panning and Jaenisch, 1998).
The brief period between fusion and PCC would thus be
best explained as a completion of late-replicating
heterochromatin in the S phase. A subsequent and later
synthesis of all of the genomic DNA synthesis could then
result in 4 S polyploidy in the absence of polar body
formation.
Aberrant centromere synthesis at these stages could
duplicate centromeres on single chromatids (Figure 4).
Unusual forms of centromere replication occur in mitotic
cells. Neocentromeres can form in latent DNA regions
devoid of the typical α satellite repeats and other sequences
in normal centromeres, and are apparently induced by
epigenetic factors (du Sart et al., 1997; Murphy and
Karpen, 1998; Wiens and Sorger, 1998). Centromeres in
higher eukaryotes, including mammals, consist of an
essential DNA core flanked by highly repeated nucleotide
sequences (Murphy and Karpen, 1995). Centromereassociated proteins in an anaphase-promoting complex
control anaphase separation by proteolysis (Jorgensen
et al., 1998). Dicentric human chromosomes, as found in
Roberstonian translocations, seemingly function by
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R.G.Edwards and H.K.Beard
Figure 4. Aberrant centromere synthesis. If premature chromosome
condensation (PCC) and incomplete (centromeric) DNA synthesis
occur (Pinto-Correia et al., 1995), chromatids would become dicentromeric. This could result in chaos, interfering with spindle formation and function, perhaps inducing conditions such as short spindles
producing anomolous divisions or even unipolar spindles leading to
chromosome doubling to 2S. Anomalies in spindle and chromosome
structure have been reported in rabbits although they seem to be
more severe with late S clones (Collas et al., 1992), and it is even
possible that nuclei are forced backwards to G1 and mitosis by maturation promoting factor (MPF). If complete DNA synthesis occurs
following centromeric synthesis as found by Pinto-Correia et al.,
(1995), this would effectively convert the nucleus to 4S DNA. Since
G1 is very short in blastomere nuclei, these comments will apply
only to nuclei from fetal or adult tissues.
inactivating one of the centromeres (Sullivan and
Schwartz, 1997). They differ in their protein constituent,
with some being typical of active centromeres.
Centromeres and other heterochromatic regions contain
repeat sequences, and are methylated and underacetylated.
They have been well studied in yeast, e.g.
Schizosaccharomyces pombe, where histone deacetylase
inhibition impairs chromosome segregation and increases
the rates of lagging and lost chromosomes by 100-fold in
mutant forms (Allshire, 1998). Whether these situations
emerge after PCC during cloning is unknown at present.
Pronuclear phenomena during in-vitro fertilization
(IVF) and intracytoplasmic sperm injection (ICSI)
related to the events of nuclear fusion
Remarkably, similar forms of abnormal development seem
to occur during normal and assisted human fertilization.
Figure 5. Diagram illustrating the timing of pronuclear and cell
cycle events. (a) Events during cloning (Pinto-Correia et al., 1995).
S1 = initial centromeric DNA synthesis; S2 = second DNA synthesis
in pronuclei; PCC = premature chromosome condensation; CS =
chromosome associating with spindle; sp = spindle formation; PN =
pronucleus formation. (b) Formation of a single nucleus in some human eggs after spermatid injection (Tesarik and Mendoza, 1996).
2PN = two pronuclei; SN = single reconsitution nucleus; SND =
single reconsitution nucleus decondensation. (c) Pronuclear history
in potential precursors of human hydatidiform moles (Edwards
et al., 1990). PN = single pronucleus; PND = pronuclear decondensation; RN = reconsitution nucleus; RND = reconstitution
nucleus decondensation; 2C = 2 cell stage.
Unipronucleate human eggs, possibly precursors of
androgenetic moles, apparently undergo two distinct
pronuclear phases, and perhaps two cycles of DNA
synthesis (Figure 5) (Edwards et al., 1990). Their single
pronucleus initially decondenses in preparation for
syngamy, but is replaced by another distinct nucleus which
forms and persists for some hours before the onset of a
normal syngamy and first cleavage. This evidence almost
suggests that hydatidiform moles are clones of the
fertilizing spermatozoon! Other unusual forms of
pronuclear reprogramming include the discovery of a
single pronucleus in human eggs after IVF. The pronucleus
was diploid 18,18,XY in four and 18,18,XX in two eggs,
indicating that they contained maternal and paternal
components (Levron et al., 1995). Synchronous nucleoplasts and unipronucleate eggs can be fused to produce
diploid human blastocysts (Azambuja et al., 1996). There
is no knowledge about DNA synthesis in these studies.
Unusual forms of pronuclear growth also occur in some
human eggs injected with spermatids during ICSI (Tesarik
and Mendoza, 1996). Among 28 eggs, 10 formed two
pronuclei which apparently fused by 16 h into a single
How identical would cloned children be?
‘syngamy nucleus’ with many nucleoli (Figure 5). Poor
activation and embryogenesis as well as the death of
human embryos in utero after spermatid injections
resembles some aspects of cloning (Tesarik et al., 1998).
Spermatid nuclei must de-differentiate and recondense,
replace protamines by histones, and adapt to the cell cycle
of the recipient oocyte (Fishel et al., 1996). Such
reprogramming events may have similarities to cloning.
Epigenetic regulators in cloning
The phenomenon of epigenesis, i.e. heritable changes in
gene function which cannot be explained by changes in
DNA sequence (Nan et al., 1998) is clearly of prime
importance in cloning. DNA methylation does not affect
the base pair sequence but is associated with gene silencing
and is now known to operate on a wide variety of genes
including developmental genes, specific allelic-inhibition
sites, transgenes and the inactive X chromosome in female
mammals. Gene silencing at these sites suppresses
transcription, and imposes position effects leading to
variegated patterns of gene expression. Fetal overgrowth in
the human Beckwith–Wiedeman syndrome could involve
imprinting defects in IGFII (Wutz et al., 1998); similar
systems could produce large calf syndrome during bovine
IVF and cloning.
Methylated sequences are not rare; they may comprise
the 35% of human DNA classes associated with
transposons and retroviruses in parasite DNA sequences
with high copy numbers (Bestor, 1998). Methylation and
imprinting may ensure that a stabilized chromatin structure
sustains transcriptional repression (Wolffe, 1998), a role
that may be more important than the regulation of
mammalian differentiation. Some genes function better in
heterchromatic sites, and perhaps in centromeric sites, than
in euchromatin. An onset of methylation at implantation is
associated with X inactivation in female mammalian
embryos, although the process may be activated by the
gene Xist and then sustained by methylation (Jaenisch
et al., 1998). Once established, this form of inactivation
could be maintained, self-perpetuating systems active over
many cell cycles, such as late DNA replication and
modifications in histone assembly which maintain dosage
compensation of X-linked genes (Panning and Jaenisch,
1998). Likewise, allele-specific repression may be invoked
by initial silencing of DNA elements and then sustained by
methylation (Brenton et al., 1998). All cells in the
trophectoderm display X inactivation, compared with 15%
in the epiblast (Jaenisch et al., 1998).
It is clear that such epigenetic modifications occurring
during cloning could play a decisive role in
799
embryogenesis. Even a seemingly simple operation such as
transferring mouse pronuclei into a different ooplasm can
impair embryonic growth (Latham and Solter, 1991), while
exchanging pronuclei, or male and female pronuclei,
evokes transcriptional repression, fetal growth retardation
and abnormal methylation of urinary proteins in offspring
even though they are diploid with paternal and maternal
genomes (Norris et al., 1990; Reik et al., 1993). The focus
on this area will be enhanced by the recent demonstration
that these epigenetic modifications can be passed to >50%
of the next generation by transmission through the male
germline in mice (Roemer et al., 1997). Indeed,
appropriate chromosomal modifications establishing
active or repressed states in gene function may be built into
chromatin as a memory of the developmental decisions
taken by individual cells, and genomic or gametic
imprinting may be a significant form of epigenetic
inheritance (Surani, 1998). It is highly probable that similar
mechanisms occur during cloning, or have to be reversed
after nuclear fusion, with considerable effects on the
resulting form of development in cloned embryos.
Practical aspects of cloning
Growth of cloned embryos
Whatever its complexities, cloning is being commercially
applied in livestock production, with hundreds of calves
born (Bondioli, 1993) and used to increase efficiency in
research studies, e.g. in pigs (Lamberson, 1994). The first
transgenic cloned lamb, Polly, carried a human gene for a
therapeutic protein (Nature, 1997) which takes cloning into
human medicine. Any clinical applications in human
embryology are precluded as long as the reasons for poor
success rates, incomplete development and high pre- and
perinatal losses in animals, particularly with late
embryonic, fetal, or adult nuclei, are not fully understood.
The success of embryonic development after cloning in
cattle has been notably poor when compared with embryos
established by IVF. Approximately 60% of those cloned
from blastomeres establish pregnancies, compared with a
pregnancy rate of 78% with IVF (Renard, 1998). These
proportions reduce to 22 and 33% respectively, at birth.
Bovine blastomere nuclei produce more blastocysts (30%)
than do germ cell or fibroblast nuclei (2 and 6%
respectively), and this is typical of the poorer results with
highly differentiated nuclei. Large calf syndrome is more
common after cloning than after IVF. Bovine embryos
cloned from embryonic nuclei produce similar numbers of
embryos in comparison with IVF, but these clones produce
more heterogeneous RNA, many of their cells die and
embryonic mortality is high, implying that reprogramming is
800
R.G.Edwards and H.K.Beard
incomplete (Heyman et al., 1995; Lavoir et al., 1997). In
contrast, comparisons of 30 datasets obtained worldwide for
several breeds of cattle showed higher rates of increased
birthweight, dystocia and perinatal death in offspring
produced by in-vitro maturation and culture techniques than
by nuclear transfer (Kruip and den Daas, 1997).
Bovine inner cell mass nuclei have been used moderately
successfully in cloning. One study yielded 34 blastocysts
transferred to 27 cows, resulting in 13 pregnancies and four
normal births (Sims and First, 1994). Another study on 948
nuclear transfers produced 5% blastocysts, and six
pregnancies from 26 transferred embryos. Two pregnancies
aborted after 60 days and two live and two stillborn calves
were delivered. A stillborn male and a liveborn female had
limb deformities, and all four died within 15 days of birth.
The average birthweight of cloned calves was significantly
greater than that of normal dairy calves (Keefer et al., 1994).
Varying nuclear/cytoplasmic ratios and cytoplasmic vesicles
characterize bovine clones established from embryonic cell
lines. Better results followed the use of small, rather than
large, cells but all embryos died by 60 days post-cloning
(Stice et al., 1996). Bovine cells derived from fetal male
gonads supported limited development to blastocyst (4%),
although none survived beyond 40 days after transfer of
three blastocysts and one morula (Delhaise et al., 1995).
Sheep blastomere nuclei allowed development to
blastocyst (35%) and established pregnancies in four out of
22 (18%) recipients (Smith and Wilmut, 1989). Embryos
cloned with nuclei from cultured cell lines failed to develop
normally, and fewer than 17% developed to blastocysts. Of
eight lambs born from 34 blastocysts transferred to seven
recipients, three died pre-birth, two died within minutes of
birth and another at 10 days (Campbell et al., 1996). The
birth of Dolly was accompanied by the death of 62%
(12/21) of the fetuses, far more than an estimated 6% after
natural mating (Wilmut et al., 1997). Two fetuses had
abnormal liver development, and losses at birth reached
12.5% (one lamb in eight), a ratio similar to the 8% loss in
commercial flocks (Nash et al., 1996).
Recloning of cloned embryos has been achieved in cattle
and mice. In cloning steps carried out over four successive
generations in cattle, the number of cloned embryos rose
from one to 54 identical morulae (Stice and Keefer, 1993).
Fusion rates declined with later generations of clones, and
developmental rates were highest with first and third
generation embryos. Several first, second and third
generation calves were born, but pregnancies were lost in
all generations as calving rates declined from 10 to 2–3%
between the first and third generations (Stice and Keefer,
1993). Recloning was also achieved using nuclei from
32-cell cow embryos (Ectors et al., 1995). Electrofusion
was followed by co-culture on feeder cells until day 6 when
the embryos were recloned and five pregnancies arose after
transfer.
Excellent success rates have been obtained with
recloning in mice. Each metaphase donor nucleus taken
from 4-cell embryos following fusion using Sendai virus
and then electrical activation was successfully cloned
(Kwon and Kono, 1996). The resulting embryos were
cultured in cytochalasin to prevent polar body expulsion
and their two pronuclei were transferred singly into
enucleated fertilized oocytes. Over 80% of the recloned
embryos developed into blastocysts, including three sets of
identical septuplets derived from a single original embryo
(Figure 2). An astonishing figure of 57% of embryos
transferred to 11 recipients developed into offspring
(Kwon and Kono, 1996). One set of identical sextuplets
and one set of identical quadruplets were obtained among
25 live offspring which were normal and fertile.
These recent studies in mice provide the brightest
outlook yet for the future applications of cloning. It is not
known what differences between species may arise and the
range has now extended to the rhesus monkey (Meng et al.,
1997). This stresses yet again that further studies are
needed in all species to resolve problems in the cloning
process including the high fetal and perinatal mortality and
the many abnormal forms. Even as this article was being
completed, outlooks on the success of cloning using nuclei
from adults has expanded to mice (Wakayama et al., 1998)
and to 28 cattle cloned from adult oviductal nuclei
(Y.Tsunoda, personal communication). The pace of work is
obviously quickening.
Variations between cloned offspring
Information on the variability of cloned offspring, a most
important outcome, is inadequate to draw any definite
conclusions. Patterns of growth, malformation and death
imply that clones are highly variable and that long-acting
genetic, developmental and epigenetic effects cause
catastrophic forms of embryonic, fetal and post-natal
development.
The highly unusual and stressful events involved in the
cloning process could impose enormous effects on the
normality of embryogenesis. Epigenesis is widely studied
in relation to methylation, position effects, switches in
identifiable gene activity, gene silencing and imprinting. It
is less well studied in relation to inherited variation and
variance analysis, although Wolffe (1998) sums up the
whole matter: ‘Gene regulation is dependent not only on
DNA sequence per se but also on the appropriate
compartmentalization of the DNA sequence. The compart-
How identical would cloned children be?
mentalization concept emphasizes the general, rather then
the local, control of gene activity’. Epigenesis has been a
major matter for embryologists, who wish to know if its
expression is regular, invariant and decisive, and how its
effects compare with gene mutation and recombination as a
source of variation (Waddington, 1956).
Sporadic information on phenotypic variation in clones
is available from several studies. Cattle cloned from the
same nuclear donor vary in size and in their colour
markings (Renard, 1998; Stice and Robl, 1988).
Birthweights ranged from 70 to 86 lb in four calves cloned
from the same donor (Sims and First, 1994) and multiple
generation recloning produced eight calves in the first
generation and one each in the second and third generations, with high variations in birthweights. Two calves
had high birthweights of 160 and 162 lb respectively,
against a mean of 84 ± 10 lb in eight others (Stice and
Keefer, 1993). In contrast, responses to parasites and
leukocytic reactions to immunological challenge probably
varied less in clones than in normal siblings (Renard,
1998).
Models have been constructed to estimate the degree of
variation expected in cloned pigs. Three traits, litter size,
days to slaughter and backfat thickness, represent wide
ranges of heritabilities with well-defined causes of
phenotypic variation. Data from four large trials were used to
partition phenotypic variation into additive and dominant
genetic, environmental and remaining non-additive genetic
and common environmental variance. Animals were
compared by range of performances comprising 95% of the
group. Littersize was predicted to range from 5.4 to 14.6
offspring per litter for clones compared with 5.2 to 14.8 for
full sibs and 5.0 to 15.0 for unrelated animals. Litter size has
a low heritability which accounts for these low inter-group
differences. Backfat thickness, with the highest heritability,
was estimated to range from 12.6 to 17.4 mm for clones
compared with 11.8 to 18.2 mm for full sibs and 11.0 to 19.0
mm for unrelated individuals (Lamberson, 1994). This
author comments that the range of performance is still wide
even in clones.
Cloned animals could reduce the number of animals
needed in various trials. The degree of reduction depends
on trait heritability. Numbers required were estimated to be
reduced by 65 and 37% using clones or full sibs for days to
slaughter, whereas savings were only 12 and 5% using
clones or full sibs for litter size in pigs (Lamberson, 1994).
In cattle, 30 clones would be expected to provide the same
information as 53 unrelated animals for highly heritable
characteristics (h2 = 0.5), where variability between
treatments is similar to variance within treatments.
Differences were few for lowly heritable traits (h2 = 0.1).
801
Additive genetic variability using clones was found to be
four times less than with unrelated animals (Colleau et al.,
1998). The value of clones over full sibs was much less,
although clones were far less variable than random
animals. Wide differences still remained among clones,
perhaps greater than those between monozygotic twins as
suggested by Lamberson’s (1994) study in which intraclass
correlations among clones were predicted as 0.15, 0.66 and
0.64 for litter size, days to slaughter and backfat thickness
in pigs. These values are generally lower than for
production traits in monozygotic twin dairy cattle, which
range from 0.83 for linear body measurements to 0.98 for
butterfat production.
Monozygotic human twins: nature’s clones
Variations in embryological origins and
development of monozygotic twins
The frequency of human monozygotic twinning was stable
at four per 1000 pregnancies before ovarian stimulation
and IVF were introduced in the 1970s. Dizygotic twinning
is inherited and has a frequency of 4–16 per 1000
pregnancies, depending on race and age (Derom et al.,
1993; Tong et al., 1997). Monozygotic twins may separate
at the 2-cell stage or at a later preimplantation stage
(Corner, 1955; Boyd and Hamilton, 1970). Occasionally,
two human embryos form within one zona pellucida, or a
blastocyst within a human blastocyst (Hardy et al., 1996).
Twinning in early cleavage stages may be a consequence of
totipotency in mammalian blastomeres (Johnson et al.,
1995). 2-cell splitting could produce dichorial and monoor dichorionic placentae (Corner, 1955).
Monozygotic twins might also separate as the diffuse
human inner cell mass divides. Rare human blastocysts
possess two separate inner cell masses within one
trophectoderm, but they might fuse as observed in mouse
blastocysts with an extra transferred inner cell mass
producing chimaeric offspring rather than twins (Gardner,
1975). Inner cell mass duplication could result in
monochorionic, diamniotic placentae (Corner, 1955), and
two-thirds of monozygotic twins are monochorionic
(Machin, 1996).
The higher rate of human dizygotic twinning since
ovarian stimulation and IVF were introduced stems from
multiple ovulations and embryo transfers. The 2–3-fold
increase in monozygotic twins could be due to anomalies in
hatching human blastocysts (Edwards et al., 1986; Derom
et al., 1993; Wenstrom et al., 1993). This is a consequence
of ovarian stimulation rather than IVF, as it occurs in
similar frequencies with both situations (Derom et al.,
1993). A hardened zona pellucida after hyperstimulation
802
R.G.Edwards and H.K.Beard
could impair hatching (Edwards and Brody, 1995),
trapping and splitting half-hatched blastocyts with inner
cell mass and trophectoderm in each half (Hardy et al.,
1996). Resulting monozygotic twins could be highly
variable, inheriting different amounts of inner cell mass
and trophectoderm, and grow quite separately or in various
membranal relationships. Since many of them could be
overlooked or misclassified as dizygotic from their
membranes, identifying DNA tests are mandatory.
Splitting at the head process stage, as in some armadillos
(Bulmer, 1970), could produce unequal or joined twins. A
monozygotic twin pair at an estimated post-ovulatory age
of 17 days had a chorionic sac containing twins, each with a
yolk sac (Heuser, 1954).
These forms of monozygotic twinning do not differ
greatly from cloning nuclei of preimplantation embryos
and may display similar developmental sequences. It is
well known that discordances can arise within
monozygotic twin pairs, involving genetic diseases,
intrauterine development, birth weights, congenital
anomalies and behaviour (for reviews, see Hall, 1996;
Machin, 1996). Machin (1996) writes ‘While it is true that
most monozygotic twins are phenotypically very similar,
there are significant numbers of monzygotic pairs who are
neither genotypically nor phenotypically ‘identical’’. Hall
(1996) comments that ‘there are clearly major exceptions
to the assumption that monozygotic twin pairs are
genetically identical.’ If cell allocations to twins differ
during early embryogenesis, discordances could arise
through somatic non-disjunction, skewed or differing
forms of X inactivation, or a non-random inactivation or
allocation of inner cells to one twin (Machin, 1996).
Chromosomal mosaicism or aneuploidy, resulting from
non-disjunction at the first cleavage division or later
explains why numerous twin sets differentially inherit
various monosomies and trisomies, and even mosaicisms
(Machin, 1996). One twin in a pair might have to wait for
the formation of a correct number of cells in its tissues, and
sharing one placenta can be dangerous for a disadvantaged
twin. A mother’s lifestyle and placental history could
emphasize such differences or invoke new ones. These
events could impose effects persisting thoughout life
(Machin, 1996), thus questioning beliefs that monozygotic
twins are genetically identical in all tissues, and explain
why neurological, immunological and structural aspects of
twin development have a genetic and a non-genetic
component (Hall, 1996).
Twinning is nevertheless less stressful compared with
cloning. A need for de-differentiation is minimal, and
stress should be slight during 2-cell separation, and not
great even during incomplete hatching. Problems unique to
monozygotic twins involve inequalities in dividing tissues
during separation, and competition within the uterus.
While these causes of discordance are absent in cloning, it
is possible that variations between uterine environments
will impose wider variations on singleton clones.
Genotypic and phenotypic variation among
monozygotic twin pairs
Monozygotic twins are invaluable for analyses of human
genetic and environmental variation, and as a model for
inheritance in clones. Most twins share sources of post-natal
variation since they are nurtured together. Others, separated
at birth, develop in separate environments until reunited
many years later, and provide invaluable data on
environmental variation distinct from genetic effects.
Twins reared apart and reunited later are a speciality of
the Minnesota Study Group (Bouchard et al., 1990a).
Their numerous analyses have permitted heritability
estimates of numerous human traits to be calculated, and
only a sample can be considered here. Even though
separate, genetic factors such as the choice of identical
environments by separated twins could cause them to grow
similarly, and such effects of sharing might be mistakenly
classified as genetic. Similar factors may emerge among
twins reared together (Bouchard et al., 1990a). This
characteristic is termed genetic-environmental covariance,
and could involve learning through experience. The
Minnesota studies assume that any resemblances between
twins reared apart are genetic, that assortive mating does
not occur, genetic effects are additive (i.e. with no
dominance or epistasis), and that genetic and environmental effects operate differently, i.e. without genotype/environment interactions.
The heritability (h2) of psychological and behavioural
traits between separated twins was 0.70, i.e. the genetic
component was 70% of the total. This estimate did not
include extreme cases since very few twins had been reared
in great poverty (Bouchard et al., 1990a). Other multiple
measures of personality, temperament, interests and social
attitudes were similar whether twins were raised together
or apart. The authors concluded that although personality
and social attitudes such as traditionalism and religiosity
could be influenced by parenting and environment, this
does not happen in most families in Western societies
(Wilson, 1983).
Two mental ability batteries, Hawaii and Comprehensive Ability, were used to measure verbal, spatial,
perceptual speed and accuracy and memory domains
among 146 twins reared apart (Bouchard et al., 1990b).
Twin sets had been separated at an average age of 0.5 years
How identical would cloned children be?
and reunited at 32.6 years, and the chance of misclassifying
monozygosity was <0.001. Intraclass correlations of
several characteristics were 0.45 and 0.48 respectively for
the two batteries of tests on monozygotic twins and 0.34
and 0.35 respectively for dizygotic twins. A biometric
model indicated an average heritability of 0.50. Most
sub-tests on the Hawaii Battery fitted the model, with
spatial ability having the largest variability and visual
memory the least. High heritabilities (h2 ≥ 0.48) were
associated with card rotations, identical pictures,
pedigrees, mental rotations and hidden patterns and lowest
heritability with things, subtraction and multiplication,
lines and dots and cubes. With the Comprehensive Ability
Battery, most variables fitted the model, verbal ability had
the highest genetic component, and genetic components
were significant in characteristics other than word fluency,
memory span and flexibility of closure. Variables with high
h2 (>0.60) included verbal, spelling, word fluency, number
and space, and those with low h2 (<0.49) included memory
span, flexibility of closure, speed of closure, peceptual
speed, and meaningful and associative memory. Many
variables
with
lowest
heritabilities displayed
non-significant or borderline P values in tests for no
genetic effects (Bouchard et al., 1990b). Comparisons
between reared-apart and reared-together twin pairs for
verbal, spatial, perceptual speed and accuracy, and
memory tests highlighted shared environment as having an
important role in determining some special mental
abilities, but a minor importance in personality and
cardiovascular domains among twin sets reared apart.
Genetic and environmental influences on motor learning
measured in monozygotic twins reared apart (Fox et al.,
1996), based on a rotary pursuit task, were fitted to a
combined genetic and environmental model. A high
heritability in performance increased with practice, and the
rate of learning was also largely inherited. Acquisition of
motor skills was strongly influenced by practice with
feedback, although non-additive genetic effects affected
the results. Practice thus decreased environmental
variation dependent on previous learning and increased a
role for inheritance of motor performance far more in
monozygotic than in dizygotic twin pairs. This observation
on reunited twins could be very relevant to clones gestated
separately and then brought together.
As in animal cloning, the variation in human clones would
largely depend on the heritability of each particular trait.
Low heritability traits would be little affected by cloning.
Much data is available concerning genetic inheritance in
man. A few examples (see Table I), illustrate the wide
differences in heritabilities resulting from a range of
conditions from single point mutations and structural
chromosomal anomalies to multifactorial inheritance and
predominantly environmental factors. It is therefore essential
to consider each feature individually. Facial features, for
example, which play such an important role in social
recognition, could be a trait of some interest with respect to
cloning. The major components of the face are derived from
cells migrating from the neural crest of the 2–3-week fetus
and anomalies arise from external environmental factors
such as alcohol, intra-uterine trauma and thalidimide, single
gene mutations such as Treacher–Collins mandibulofacial
dysostosis and multifactorial inheritance including cleft lip
(with or without cleft palate) as shown in Table I (Kaye,
1981). Thus for a number of different traits it would be
possible to predict the variation expected between clones as
between monozygotic twins and, therefore, society should
analyse and clarify the nature of its abhorence for the concept
of cloning in humans.
Table I. Wide differences in heritability (h2) estimates for some chosen human conditionsa
Condition
h2
Comment
Reference
Appendicitis
0.56
Polygenic or multifactorial model based on
Basta et al. (1990)
80 families with histopathological acute
appendicitis and matched surgical controls
Breast cancer
0.3–0.4
Analysis of 45 monozygotic twins
Holm et al. (1980)
and 77 dizygotic twins
Cleft lip
Duodenal ulcer
0.77–0.97
Comparison of Danish and Japanese populations
Chung et al. (1986)
0.91
Prominent history in two large families
Rotter et al. (1982)
Average heritabilities of δ, θ, α and β
Van Beijsterveldt
frequencies (76, 89, 89, 86% respectively)
et al. (1996)
0.74 dominance
Electroencephalogram
aData
0.76–0.86
803
from Mendelian Inheritance in Man, V.McKusick, online version http://www.ncbi.nlm.nih.gov/omim/.
804
R.G.Edwards and H.K.Beard
Examples of discordances within monozygotic
twin sets
Unusual differences and unusual similarities among twin
sets often indicate de novo embryological events or rare
genetic differences. Discordance for juvenile chronic
myelogeneous leukaemia (JCML) in a twin set confirmed
by DNA analyses arose through their joint inheritance of a
de-novo deletion in the pericentric region of chromosome
7, a known cause of this disorder (Najfield et al., 1997).
The more severely affected twin could have inherited more
cells carrying a mutation arising before uneven splitting of
the original embryo. Shared environments and common
genetic factors of monozygotic twin pairs could explain
similarities in within-pair spontaneous mutation frequency
of hypoxanthine–guanine phosphoribosyltransferase
(hprt) in circulating T-lymphocytes, when compared with
the wider variations between pairs (Curry et al., 1997).
Environmental influences may evoke discordances. One
twin in a monozygotic set checked by DNA analyses had
hand abnormalities and hearing loss in oculo-oto-radial
syndrome whereas the other had strabismus only, perhaps
as a consequence of differing epigenetic or environmental
factors (Elcioglu and Berry, 1997). Discordancies for
isolated congenital complete heart block within two twin
sets checked by DNA analyses, arose soon after birth in one
twin and at 3 years in the other, despite their identical
genetics and common environmental exposure to anti-Ro
antibody (Cooley et al., 1997). Leptin concentrations were
almost 4-fold higher in the obese twin among 23 discordant
monozygotic twin pairs assessed for fasting concentrations
of plasma leptin and distribution of visceral and
subcutaneous fat (Ronnemaa et al., 1997). Intrapair
differences correlated with corresponding variations in the
percentage of body fat in women (r = 0.73, P = 0.003) but
not in men, and with differences in visceral fat area in men
and women, and had arisen independently of genetic
background (Ronnemaa et al., 1997). Among 53
monozygotic twin pairs reared apart, with a mean body
mass index (BMI) of 24.2 ± 4.7, heritability was 0.79 for all
twins, 0.63 for Finnish twins in the sample, 0.73 for
Japanese twins and 0.85 for ‘archival’ twins (Allison et al.,
1996). One-half or more of the variance in heritability not
accounted for by covariates was genetic. In two of 14
monozygotic twins sets discordant for endometriosis when
adult, one twin reached stage II–IV of the disease whereas
the other was unaffected (Hadfield et al., 1997); secondary
effects such as uneven splitting could explain the discordance.
Discordances for characters displaying lateral asymmetry include handedness, which is well known to diverge
in monozygotic pairs. More left than right handedness
among 1616 monozygotic twins was independent of chorion type and zygosity (Derom et al., 1996). Families likely
to have monozygotic twins display a tendency not to use
the right hand. Machin (1996) considers that an abnormal
blastogenesis could explain discordances for lateral body
wall defects and other conditions.
Some genetic differences could arise between monozygotic twin pairs as in the X-linked condition, Aicardi syndrome, a condition involving agenesis of the corpus
callosum, seizures and anomalies of the chorioretina.
Slightly more methylated sequencies flanking the DxS225
locus in the afflicted twin of a discordant dichorionic pair
may have arisen through a post-zygotic mutation and unequal distribution of mutated cells, skewed X inactivation,
germ cell mosaicism or other X-linked disorders (Costa
et al., 1997). Post-zygotic de-novo mutations within two
monozygotic twin pairs may have caused discordancies for
tuberous sclerosis and a form of muscular dystrophy (Brilliant et al., 1990; Tawil et al., 1993). One non-afflicted
monozygotic twin in a 47 year old pair did not carry a
familial BRCA1 mutation nor suffer from repeated cancers
as did all other family members (Diez et al., 1997). The
discordance could not be traced to age of menarche, contraceptive use, pregnancy, hormone therapies, smoking or
drinking, environment, exercise, habits or diet. Discordance for the Schimmelpenning–Feuersstein–Mims syndrome (SFM) was attributed to a post-zygotic lethal
mutation in one monozygotic twin who suffered from
childhood epilepsy and developmental and mental retardation (Schworm et al., 1996).
Genetic differences involved higher concentrations of
two cell recognition molecules (CRMs) and neural-cell
adhesion molecule (N-CAM) and lower values of antigen
L1 in cerebrospinal fluid in affected twins within monozygotic sets discordant for schizophrenia (Poltorak et al.,
1997). N-CAM and L1 in cell membranes regulate cell/
environment contacts essential for neural development and
synapsis formation. Genomic mutations or imprinting
might cause discordances preceding the onset of disease, or
be associated with its pathological course.
A ‘non-shared environment’ can involve wide differences between children within a family as in members of
the same pair among 93 monozygotic sets aged 10–18
years, chosen in order to relate the effects of shared and
non-shared influences on children of the same genotype
(Pike et al., 1996). Parental negativity and the twins’ antisocial behaviour were related perhaps by differences in
parental treatment, although effects of twins behaviour on
parents, or other factors, could not be excluded. Carried out
on middle-class children, this study requires confirmation
How identical would cloned children be?
by similar work on other classes, or on extrafamilial influences such as peers. A delay in talking and problems
with relationships outside those with their twin characterize the difficulty of some monozygotic pairs in establishing
their identity (Bryan, 1998). Length of gestation influences
tempremental discordancy within discordant monozygotic
pairs developing to full-term, but not those born pre-term
(Riese, 1996). Pre-term twins also grow more alike in emotionality, and full-term discordant twins soon overcome
adverse uterine influences on early behaviour.
According to Machin (1996), such considerable physical, emotional and psychological divergencies among
monozygotic twin pairs imply that ‘identical’ should be
replaced by ‘monozygotic’. Hall (1996) concurs, suggesting that many twin discordances arise through varying intrauterine environments, different cell allocations to each
twin, different placental vascular supply and stochastic developmental differences. She also infers that some discordances may be actually cause twinning. The twinning
process must occur independently of specific embryological characteristics if monozygotic sets are to be acceptable
models of cloning. In other words, discordance within
monozygotic pairs must be a consequence and not a cause
of twinning. Although still theoretical, the formation of
two or more cell clones in an inner cell mass could stimulate twinning or variations in the formation and vascularization of the placenta.
How closely would cloned children resemble
their parents and each other?
This fundamental question can be answered only by
evidence from human cloning. Animal studies provide
some clues, since all eutherian mammals may share the
same form of developmental genetic regulation (Edwards
and Beard, 1997). Species differences in implantation, in
organogenesis, and in numbers of gestated fetuses
complicate comparisons with humans. Clones could vary if
derived from nuclei of different tissues of the donor, with
varying post-fertilization ages or differing numbers of
previous cell divisions. At present, high frequencies of
anomalies and intrauterine or post-natal deaths
characterize animal clones produced from fetal or adult
nuclei, unlike those derived from blastomere nuclei.
Variations in growth may also be imposed by incomplete
activation of cloned nuclei, differing components in
recipient ooplasm, and genetic differences in maternal
transcripts and mitochondria. Outcomes not unlike those of
cloning characterize other micromanipulations on
mammalian eggs, implying a common etiology of
disorders due to stress or other underlying factors.
805
Human clones from the same donor are likely to vary
considerably. They will have no ‘mother’ for gestation, and
will grow in surrogates, as already happens for the
gestation of IVF fetuses for commissioning couples,
undoubtedly offering highly diverse uterine environments.
Physiological variations within the uterus, caused by
smoking, drinking, drugs and infections, will distort
normal growth patterns in fetuses as reported in mothers
taking cocaine (Rizk et al., 1997). The site of implantation,
degree of vascularity and presence of structural or other
disorders will also dictate the birth characteristics of
clones, including variations in their multigenic
characteristics. Divergences in jawbone structure among
newborn mice were initially ascribed to mutations induced
by embryo cryopreservation, but are now suspected to be
experimental or statistical artefacts (Testart, 1998).
Disorders imposed on human embryos during uterine or
post-partum life can affect the child and adult for years to
come. Nor can there be much doubt that uterine and social
environments for cloned children will differ from those
experienced by their nuclear donor.
Monozygotic twins identify some of the variations likely
to arise in clones. They are suitable models since most
originate at the 1–64-cell stages (1–6 cleavages), which are
the embryonic stages widely used for cloning. Embryo
splitting might expose twin embryos to similar forms of
stress as in cloning. Discordancies in genetic,
developmental and psychological characteristics within
monozygotic pairs must be caused by epigenesis or
environment. Studies on monozygotic twins reared apart
have proved extremely valuable, since their post-natal lives
are not shared, so their degrees of environmental variation
arising after birth can be calculated. Discordances within
monozygotic pairs can lessen among those reunited at a
later age, and among those who are discordant at birth
becoming more concordant post-natally.
All this evidence suggests that clones will be at least as
divergent as monozygotic twins, who are accepted without
question in society. The one medical group to propose a
reduction in monozygotic twinning by banning fertility
drugs (Derom et al., 1993), did so on obstetric rather than
psychological or behavioural grounds. Most twins live
happily within a family circle, and many consider
themselves highly fortunate in having such a close friend
throughout life. Many dizygotic twins feel the same.
Personality and other difficulties seem to stem partially
from their environment within the family (Bryan, 1998),
but none are of such an order as to question their existence
or happiness.
Could clones be accepted in society, just like twins?
Widely-quoted objectors stress that human personality must
806
R.G.Edwards and H.K.Beard
be inherited absolutely freely and randomly, since it is a
fundamental right never to be jeopardized by the whim of its
parents. Yet society already imposes restrictions to prevent
consanguinity and by restricting the number of births from
sperm donors. Parents already choose particular
characteristics in their children. Female fetuses are aborted or
newborn girls killed in some societies for various complex
reasons about the value of boys. Embryos are selected, and
fetuses aborted, because they inherit a particular genetic trait.
Indeed, science itself is demeaned by a familiar process,
starting with the identification of a new disease gene, followed
by the consequential abortion of fetuses carrying it. A
well-known sperm bank (apparently in California!) stores
spermatozoa from Nobel laureates, suggesting that both
donors and recipients of such gametes approve this attempt to
‘improve’ the human race. What, then, is so special about
cloning? An American lawyer, Robertson (1997), stresses that
cloning is less intrusive than fetal gene therapy and that cloned
children will be loved for themselves, have all rights of
existing persons, be no more the property of the
commissioning couple than any other child and will be
unrecognizable from other children. Desires to clone emerge
in forces as diverse as love for a recently-dead child or lust of
power-hungry despots to ensure their succession. The first
example is a product of love, the second of power. Should a
sterile couple be denied the right to clone a donor of their
choice because of society’s fears and therefore be compelled
to accept donor embryos unknown and genetically unrelated
to either of them? Cloning could be desirable or objectionable,
and perhaps more desirable if the children display differences
wider than monozygotic twins.
Cloning from embryonic nuclei does not hold the same
moral fears as adult cloning. It involves neither ‘carbon
copies’ nor the many anomalies associated with fetal or adult
nuclei. Cloning monozygotic twins from blastomeres
provides an origin very similar to the origins of most
naturally-occurring monozygotic twins. Viewed from this
perspective, blastomere cloning offers a procedure to increase
numbers of available embryos in infertility treatments without
imposing a drastic departure from natural embryological
events. Although limited to the use of inbred strains, possibly
restricting epigenetic variation, a successful model already
exists in mice (Kwon and Kono, 1996). Re-cloning existing
blastomere clones, also highly successful in mice, is more
problematic. It opens dangers of storing many cloned
embryos while a few are transferred to test their social and
personal attributes. Unwelcome aspects of cloning, such as
this form of progeny testing, could be controlled by law, just
as the number of offspring permitted from a semen sample is
restricted legally.
Is the inheritance of a highly variable genetic trait due to
gene recombination and mutation an inalienable and essential
right that is threatened by cloning? Monozygotic twins do not
think so. We have been taught throughout our lives that man
(like animals and plants) is a product of a highly variable
genetic inheritance, modified in an ever-changing
environment. This has been true in the past, but need not be so
in the future. Mankind’s gene pool and its variability has been
based on family structures and infant survival rates, processes
frozen by the spread of contraception, medical care and
infertility treatments. New forms of variation could become
essential one far-off day. The significance of variation as an
essential evolutionary human characteristic has stimulated
debate among geneticists, behaviourists and sociobiologists.
We initially concurred with, but now question, a recent value
judgement about the importance of modern society from
Bouchard et al., (1990a): ‘A human species whose members
did not vary genetically with respect to significant cognitive
and motivational attributes, and who were uniformly average
by current standards, would have created a very different
society to the one we know. Modern society not only
augments the influence of genotype on behavioural
variability…but permits this variability to reciprocally
contribute to the rapid pace of cultural change’. Is this
statement correct, and does it apply to societies with a high
level of modern contraception? How significant is epigenesis
to our quantitative inheritance and variation? Dolly seems to
be healthy and happy, even if her birth was at the expense of
an enormous mortality rate. Polly and recently-born calves
carry genes inserted for human needs. Genes could be inserted
to improve the life of the recipient, e.g. for a long life or to
introduce new forms of variation. Such procedures could
prove of enormous potential significance and value to future
human generations, and cannot be dismissed too easily.
Furthermore, fears of cloning for spare-parts surgery for
donors could be unfounded, outdated by the clinical
application of cell de-differentiation in vitro and reconstructive surgery. Within a few years, let us hope that desired cell
lines will be available for self-grafting by de-differentiating
cells taken from our own tissues.
Conclusions
Society always reacts unfavourably to new concepts,
especially in reproduction. Fears of the unknown raised about
contraception, IVF, embryo cyopreservation, surrogacy,
embryo donation, genetic engineering and other matters have
proved to be largely unfounded, and without earth-shattering
consequences. Indeed, most of them have already increased
the overall sum of human happiness and are now practised
How identical would cloned children be?
worldwide. Sadly, recent consultative documents from
various European authorities revert to an earlier and more
restrictive age (Shenfield, 1998). Moral and philosophical
strictures against cloning are raised again, as a threat to
uniqueness and ‘autonomy and dignity’ of each human being
which creates an ‘intolerable lowering of a person to the status
of an object’ and introduces ‘new kinds of slavery’ by ‘the
creation of human varieties’ (Boué, 1998). The same thing
was said about contraception, abortion and IVF. What
scientific rationale lies behind such statements? Hottois
(1998), a philosopher himself, seems to be highly
unimpressed on philosophical grounds by these arguments.
Parents often interfere with the fundamental interests of their
children. Their self-interest can often outweigh those of the
child during adoption, genetic diagnosis of embryos and
fetuses, gamete donation and surrogacy. Only one progressive
European organization, the European Society of Human
Reproduction and Embryology, (ESHRE), dissented from
general criticisms of cloning as it appealed to members to
delay attempting it for 5 years until its ethical and biological
implications were clearer (ESHRE, 1998). We agree with this
stance, and with a US approach requesting a 5 year ban and a
re-evaluation at the end of this time (National Bioethics
Advisory Commission, 1997). They wrote that ‘More
speculative psychological harms to the child, and effects on
the moral, religious and cultural values of society may be
enough to justify continued prohibitions in the future, but
more time is needed for discussion and evaluation of these
concerns’.
We suggest that human cloning is premature, until animal
studies reveal its potential and disadvantages, and until
variance among clones is assessed in detail. Before cloning is
accepted, it would be more important to know much more
about epigenesis as a source of human variation. A delay of 5
years should be sufficient for some decisions on cloning, e.g.
whether to duplicate 2- or 4-cell embryos to produce
monozygotic twins for sterile couples with only a single
embryo in vitro. We find no scientific reasons for an outright
ban, nor any rational basis for some of the objections. We
welcome an open approach to clarify potential benefits to
particular individuals and couples, and look forward to
understanding the long-term effects of cloning.
Acknowledgements
We wish to thank Dr Jean-Paul Renard for his very helpful
criticisms of our paper and Dr W.R.Lamberson for helpful
comments on the section Practical Aspects of Cloning.
807
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