Download Reconstitution of gametes for assisted reproduction U.Eichenlaub

Reconstitution of gametes for assisted reproduction
oocyte possesses such ability. It remains to be determined
whether germinal vesicle (GV) stage oocytes can fully erase
and restore genomic imprinting information. A recent report
suggested that normal chromosome segregation may occur in
GV stage oocytes (Palermo et al., 2002b). Another study
reported that haploidization does not occur with GV stage
oocytes (Fulka et al., 2002). Because genomic imprints
appear to be established very early during oogenesis (Obata
and Kono, 2002), it is unlikely that fully grown GV stage
oocytes would retain the capacity to erase and restore genomic
imprints. Even if this were to occur, it is unreasonable to
propose that a substitute paternal gamete genome could be
obtained from an oocyte, as has been suggested (LachamKaplan et al., 2001).
Given the fundamental limitations of chromosomal segregation and genomic imprinting, the notion of using the MII
oocyte to drive haploidization of a somatic cell genome and
thereby obtain a substitute for authentic gametes is illconceived and untenable (<7 3 10±13 overall). These two
limitations together relegate this proposed methodology to the
realm of the fantastic. This is not a matter of controversy, but
merely a basic aspect of mammalian reproduction.
Reconstitution of gametes for assisted
reproduction
Acknowledgements
There are basically two major problems in the genesis of
`cloned' gametes in mammals, which have not been addressed
in the debate article by Tesarik (2002). There is no adequate
discussion on the mechanisms providing for high ®delity of
chromosome segregation in mitosis and meiosis, and for proper
imprinting in construction of `reconstituted gametes'. The
debate article is uncritical with respect to the currently
insuf®cient database and the incomplete documentation of
results.
The reductional segregation of parental chromosomes,
which have been originally derived from the father and
mother, usually requires a physical connection between
homologous chromosomes. Physical association is mediated
by the presence of at least one chiasma at a site of genetic
exchange on all chromosomes in male and female meiosis in
mammals and cohesion between sister chromatids (also termed
monads) of homologues (also termed dyads or univalents)
within each bivalent. Failures in recombination greatly
increase the risk for random segregation of univalents
(Hassold and Hunt, 2001; Nasmyth, 2001; Eichenlaub-Ritter,
2003). Properties built into the chromosomes and not the
cytoplasm or spindles determine the behaviour of chromosomes at meiosis, such that transfer of single bivalents from a
meiosis I spindle into a meiosis II spindle by micromanipulation, will result in separation of homologues (dyads) and not
the chromatids (monads). Chromosomes from a meiosis II
spindle placed into a ®rst meiotic spindle segregate sister
chromatids (monads) whereas bivalents in the same spindle
separate homologues (Paliulis and Nicklas, 2000).
In normal chiasmate meiosis II of mammals it is essential
that sister chromatids of the haploid, replicated set of
metaphase II (MII) chromosomes remain physically attached
at their centromeres until anaphase II to mediate orientation of
centromeres to the two opposite spindle poles and high ®delity
Research in the authors' laboratories was supported in part by grants
from The Ministry of Education, Culture, Sports, Science, and
Technology of Japan (H.T.), the Harold Castle and the Victoria Geist
Foundation (R.Y.), and the National Institutes of Health, NIH/NICHD
HD38381.
References
Fulka, J., Jr, Martinez, F., Tepla, O., Mrazek, M. and Tesarik, J. (2002)
Somatic and embryonic cell nucleus transfer into intact and enucleated
immature mouse oocytes. Hum. Reprod., 17, 2160±2164.
Kaneko, M., Takeuchi, T., Veek, L. L., Rosenwaks, Z. and Palermo, G. D.
(2001) Haploidization enhancement to manufacture human oocytes. Hum.
Reprod., 16 (Suppl 1), 4±5.
Lacham-Kaplan, O., Daniels, R. and Trounson, A. (2001) Fertilization of
mouse oocytes using somatic cells as male germ cells. Reprod. Biomed.
Online, 2, 203±209.
Obata, Y. and Kono, T. (2002) Maternal primary imprinting is established at a
speci®c time for each gene throughout oocyte growth. J. Biol. Chem., 277,
5285±5289.
Palermo, G.D., Takeuchi, T. and Rosenwaks, Z. (2002a) Oocyte-induced
haploidization. Reprod. Biomed. Online, 4, 237±242.
Palermo, G.D., Takeuchi, T. and Rosenwaks, Z. (2002b) Technical approaches
to correction of oocyte aneuploidy. Hum. Reprod., 17, 2165±2173.
Takeuchi, T., Kaneko, M., Veek, L.L., Rosenwaks, Z. and Palermo, G.D.
(2001) Creation of viable human oocytes using diploid somatic nuclei. Are
we there yet? Hum. Reprod., 16, 160±164.
Tateno, H., Akutsu, H., Kamiguchi, Y., Latham, K.E. and Yanagimachi, R.
(2003) Inability of mature oocytes to create functional haploid genomes
from somatic cell nuclei. Fertil. Steril., 79, 216±218.
Tesarik, J. (2002) Reproductive semi-cloning respecting biparental origin:
Embryos from syngamy between a gamete and a haploidized somatic cell.
Hum. Reprod., 17, 1933±1937.
Tesarik, J., Nagy, Z.P., Sousa, M., Mendoza, C. and Abdelmassih, R. (2001)
Fertilizable oocytes reconstructed from patient's somatic cell nuclei and
donor ooplasts. Reprod. Biomed. Online, 2, 160±164.
U.Eichenlaub-Ritter
Universitat Bielefeld, Fakultat Biologie, Gentechnologie/
Mikrobiologie, Postfach 100131, D-33501, Bielefeld, Germany.
E-mail: [email protected]
There are basically two major problems in the genesis
of `cloned' gametes in mammals, which have not been
addressed in the original debate article. There is no adequate discussion on the mechanisms providing for high
®delity of chromosome segregation in mitosis and meiosis, and for proper imprinting in construction of `reconstituted gametes'. The original debate article is
uncritical with respect to the currently insuf®cient database and the incomplete documentation of results.
Key words: assisted reproduction/cloning/gamete reconstitution/
haploidization
473
U.Eichenlaub-Ritter
of chromosome segregation at second meiosis (Nasmyth,
2001). G1 chromatin of a diploid cell does not comprise a
single set of MII chromosomes, each with two sister
chromatids (one gonosomal and 22 autosomal MII chromosomes), but instead, consist of two sets of physically
unattached, unreplicated chromosomes (in human 44 autosomal and two gonosomal monads). There is no cohesion
between each of the parental pairs of homologous chromosomes at centromeres under these conditions. One would
therefore expect that chromosome segregation is entirely
random when forcing such `partner-less' chromosomes into
an anaphase. Chances of obtaining a haploid complement in
terms of correct number and chromosomal complement are
therefore extremely low. Any chromosome might randomly
attach to spindle ®bres and a spindle pole, and migrate to a pole
irrespective of the behaviour of the other parental copy.
Although several species have evolved complex mechanisms to ensure that chromosomes may segregate in absence of
recombination (e.g. in Drosophila males; Wolf, 1994), a
number of past and recent studies clearly demonstrated that it is
not only the presence but also the number and distribution of
chiasmata, which in¯uence proper chromosome segregation at
meiosis in mammals (Hassold and Hunt, 2001; Yuan et al.,
2002; Eichenlaub-Ritter, 2003). Precocious separation of sister
chromatids prior to anaphase II is one of the features associated
with maternal ageing, which is discussed in the predisposition
to random chromosome segregation in human oocytes and
implantation failure, trisomy, and spontaneous abortion
(Wolstenholme and Angell, 2000; Hassold and Hunt, 2001;
Sandalinas et al., 2002; Eichenlaub-Ritter, 2003).
Still, it is amazing that the few oocytes or embryos derived
from reconstituted gametes, which have been analysed in more
detail so far, had often a normal or near normal chromosome
number (e.g. 20 mouse or 23 human chromosomes respectively; Lacham-Kaplan et al., 2001; Palermo et al., 2000a,b).
Such observations have apparently contributed to the enthusiasm and unwarranted hopes associated with the technique.
How can we explain this unexpected behaviour in view of all
the genetic data giving clear evidence that recombination is an
essential feature for normal reductional chromosome segregation during germ-cell formation in mammals? Ooplasm has an
amazing capacity to organize bipolar spindles, even in the
absence of chromosomes, which requires expression of
microtubule motor proteins, tubulin, and cell extracts with
active maturation promotion factor and cytostatic factor (Heald
et al., 1996; Walczak et al, 1998; for further references see:
Eichenlaub-Ritter, 2003). Back-up mechanisms, which also
require the activity of motor proteins, are at the basis of the
unexpectedly high, non-random probability of pairs of nonexchange univalent chromosomes without a chiasma to
segregate to opposite rather than the same spindle pole during
oogenesis in some species (Karpen et al., 1996). However,
absence of bivalents impairs the formation of a normal bipolar
spindle in mammalian ooplasm entirely (Woods et al., 1999).
This may contribute to aberrant spindles and uncontrolled
chromosome segregation in reconstituted oocytes (Fulka et al.,
2002).
474
One would expect that chances of faithful segregation of the
paternally- and maternally-derived chromosomes are therefore
minute. The few FISH studies with a limited number of
chromosome-speci®c probes suggest that some of the `reconstituted gametes' segregated some of the chromosomes from
their parental second copy during division in a non-random
fashion (Palermo et al., 2002a,b). In one case what appeared by
Giemsa staining to be a haploid set of chromosomes was
obtained (Palermo et al., 2002b). However, very few cells were
properly analysed, and analysis was generally performed at a
late stage after nuclear transfer. Here, much more basic
research is required to (i) clearly de®ne the stage of the cell
cycle of the somatic nucleus used for transfer; (ii) to follow
spindle formation and behaviour of individual chromosomes at
prometaphase and anaphase after reconstitution; (iii) to determine whether `haploidized' chromatin can replicate during ®rst
mitotic S-phase after fertilization and (iv) to characterize the
number and identity of the replicated set of chromosomes
derived from the reconstituted maternal as well as the paternal
pronucleus individually. It is doubtful whether the expected
results warrant all the effort.
This is of special relevance when considering the issue of
imprinting, which is so vital for normal development. We know
that the mammalian oocyte acquires maturational and developmental competence in a gradual, stepwise fashion during the
entire period of oocyte growth and folliculogenesis (Obata and
Kono, 2002). During this period it is not only the accumulation
of cytoplasmic components but also imprinting processes
associated with chromatin remodelling and modi®cation of
DNA and chromosomal proteins, which are required to obtain a
gamete with high developmental potential. It is doubtful
whether cytoplasm of a fully grown oocyte can establish the
gender-speci®c maternal imprinting, which is usually occurring throughout the growth phase, and even more improbable, a
male-speci®c imprinting pattern to reconstitute a male
pronucleus (Lacham-Kaplan et al., 2001). Even when each of
the originally paternally and maternally-derived chromosomes
of a somatic nucleus would segregate only one copy into the
oocyte in absence of meiosis and recombination, for each
chromosome there is a 50% chance that it contains the wrong
imprint, so that after fertilization it causes bi-allelic expression
or repression of imprinted regions similar to that which gives
rise to congenital abnormalities and genetic disease in cases of
uniparental disomy (e.g. Prader-Willi or Angelman syndrome).
In addition to all these risks, introduction of a foreign nucleus
into cytoplasm with host mitochondria is still under debate
since the consequences of heteroplasmy and potentially
disturbed interactions between donor mitochondria and
cytoplasm, and the nuclear compartment from another genetically distinct individual are unknown (St John, 2002).
Accordingly, risks for implantation failures, congenital
abnormalities and inheritable disease early or late in life of
an individual derived from a fertilized, reconstituted gamete
are unpredictable. In view of all these considerations and
uncertainties, gamete reconstitution by nuclear transfer without
meiosis should not be discussed in the context of assisted
reproduction, since it may raise unwarranted hopes in infertile
couples and instigate studies with little expectation of success.
Reconstitution of gametes for assisted reproduction
I consider that it might be worthwhile to perform further basic
research in appropriate animal systems in well-designed and
controlled studies using nuclear transfer technology and
ooplasm. For instance, to introduce nuclei from different
sources into ooplasm may be an interesting approach to learn
more about the organization and regulation of chromatin and
the role and consequences of polarized chromatin organization
within cells for chromosome behaviour (Tanabe et al., 2002).
Although this may not have a direct impact on assisted
reproduction it might enhance our general understanding of the
molecular biology of cells.
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Somatic and embryonic cell nucleus transfer into intact and enucleated
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