Universitat Bielefeld, Fakultat Biologie, Gentechnologie/Mikrobiologie, Postfach 100131, D-33501, Bielefeld, Germany. E-mail: eiri@biologie.uni-bielefeld.de
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Abstract |
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Key words: assisted reproduction/cloning/gamete reconstitution/haploidization
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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 first 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 fidelity 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 fibres 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 influence 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 non-exchange 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
).
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-specific 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 define 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 first 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 modification 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-specific maternal imprinting, which is usually occurring throughout the growth phase, and even more improbable, a male-specific 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. 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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References |
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