Centre for Reproductive Medicine, Department of Obstetrics & Gynaecology, St. George's Hospital Medical School, Cranmer Terrace, University of London,London SW17 ORE, UK
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Abstract |
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Key words: abnormal paternal chromosomes/blastocyst culture/genomic imprinting/ICSI
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Introduction |
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Transformation of the transcriptionally silent sperm head to a male pronucleus consists of a series of macromolecular events: sperm chromosome decondensation, release of protamines, DNA repair, chromosomal remodelling, assembly of organelles and a nuclear envelope around the reprogrammed haploid chromosomes (Collas, 1998; Collas and Poccia, 1998
). All these events are accomplished by molecular chaperones, histones, non-histone structural proteins, DNA-repair enzymes and factors transiently accumulated in the ooplasm. Indeed, this maternal stockpile, in amphibians and flies, is so extensive that the zygote can replicate several thousand times independently of parental gene activation (Newport, 1987
; Berrios and Avilion, 1990
). In humans and higher mammals, successful male pronuclear assembly and fertilization are therefore largely determined by the quality of the oocyte cytoplasm (chromosome decondensation, DNA repair, demethylation and remodelling factors) rather than by the integrity of the paternal genome. Moreover, a direct correlation of sperm DNA damage with failed human fertilization remained debatable (Janny and Ménézo, 1994
; Sakkas et al., 1996
; Hammadeh et al., 1998
; Twigg et al., 1998b
), possibly because the non-genetic factors contributed by the spermatozoa were not considered in these studies. Activation of endogenous metabolic pathways producing reactive oxygen species (ROS) is the major cause of human sperm DNA damage (Aitken et al., 1998
; Twigg et al., 1998a
). ROS, however, can simultaneously damage sperm membrane lipoproteins and the proximal centriole, which are essential for optimum chromosome decondensation and maternal chromosome separation prior to female pronuclear assembly respectively (unpublished data; Simerly et al., 1995; Sathananthan et al., 1996; Van Blerkom, 1996).
In order to evaluate the effect of sperm chromosomal abnormalities on pre-implantation development, it is imperative to reconcile what specific contribution the parental genome could possibly have in blastocyst formation. This has been studied extensively in the mouse system where parthenogenetic, gynogenetic and androgenetic embryos can be readily created and cultivated (Barton et al., 1984; McGrath and Solter, 1984
; Surani et al., 1986
). These studies have revealed that parthenogenetically activated haploid oocytes (diploidized by preventing first cleavage division), diploid gynogenones (maternal chromosomes) and androgenones (paternal chromosomes), could all (except XX and YY androgenones because paternal X is inactivated in blastocysts by imprinting and lack of X-linked gene products, respectively) produce morphologically healthy blastocysts (see Figure 1
). However, the efficiency of androgenetic blastocyst development was invariably poor compared with those embryos derived from maternal only (parthenogenones and gynogenones) and biparental (normal) genomic contributions. Nevertheless, analysis of the newly synthesized proteins as a measure of genomic activation (24-cell stage) revealed no qualitative differences between androgenones and parthenogenones or gynogenones. Therefore, development of the fertilized oocyte to the blastocyst stage is relatively independent of the parental genotype. Additionally, the maternal genome is more competent for embryonic activation and blastocyst development compared to its paternal counterparts. This is important given the high frequency (2550%) of aneuploidy in human oocytes (Martini et al., 1997
). Failure of blastocyst development from 24-cell embryos following ICSI most possibly arises from inadequate genomic reprogramming (for instance genome-wide demethylation) necessary for subsequent gene activation, cleavage and cell determination (Walsh and Bestor, 1999
). This is a function of the oocyte cytoplasm as amply evidenced in animal nuclear (for a review, see Kikyo and Wolffe, 2000) and human cytoplasmic (Cohen et al., 1998
) transfer studies.
The consequences of inheriting abnormal paternal chromosomes are most likely to be manifested during implantation and post-implantation development. Two lines of experimental evidence support this notion. Firstly, the Harwell group (Mammalian Genetics Unit, MRC, Oxford, UK) made extensive use of mouse mutants, carrying Robertsonian and reciprocal translocations, by creating animals uniparentally disomic for specific regions of chromosomes (Pat.Dp or Mat.Dp). These studies showed that parental genes in at least 15 different regions (each region spanning several megabases of DNA) of 10 chromosomes (nos. 2, 6, 7, 9, 11, 12, 14, 17, 18 and 19) were functionally heterozygous (Cattanach and Beechey, 1998). Uniparental duplication of these chromosomal regions, with paternal or maternal deficiency, leads to a variety of developmental disorders including early or late embryonic lethality, overgrowth/reduced placenta, pre-, post-natal and fetal growth retardation, neurological abnormalities, etc (Beechey and Cattanach, 2000
). Secondly, nuclear transplantation studies led to the discovery of non-Mendelian functional inheritance (epigenesis) of parental chromosomes. Such parentally inherited epigenetic programs dramatically influence embryogenesis after implantation. In general, paternal and maternal genomes contribute to the proliferation of extra-embryonic tissues and embryo proper, respectively (McGrath and Solter, 1984
; Surani et al., 1986
).
The functional inequality of haploid genomes is due to imprinting of specific chromosomal loci/genes during gametogenesis when the parental alleles are physically separated (Tucker et al., 1996; Banerjee and Smallwood, 1998
; Surani, 1998
; Tilghman, 1999
). The imprints are established in the gamete, passed on to the zygote and must withstand embryonic activation (24-cell stage) for the successful development of the embryo to term (Surani et al., 1986
). Unlike the mouse, data on the developmental consequences due to loss of imprinting (LOI) or loss of heterozygosity (LOH) in human are far less comprehensive (see Table I
; Ledbetter and Engel, 1995). The most extensively studied chromosomes are 11 (11p15) and 15 (15q11-13) where duplication, translocation or deletion of these regions led to paediatric disorders, e.g. BeckwithWiedemann and PraderWilli/Angelman syndromes. The functional imprints of genes located at human chromosome 11p15.5 (IGF2, H19, p57kip2 and KvLQT genes) or those controlled by imprinting centres at 15q11-13, are generally established following implantation of the embryo (Latham et al., 1993
; Szabo and Mann, 1995
; Walsh and Bestor, 1999
). Paternal chromosomal damage, or failure to maintain the imprinting markings during early activation, could lead to aberrant development of the embryo. For example, paternal or maternal duplication of the Igf-II/H19 genes located at the distal end of mouse chromosome 7 (dist.7) and simultaneous maternal or paternal deficiencies, respectively, severely affects embryo development. Embryos paternally disomic for the distal 7 (Pat. Dp.Dist.7) die within 710 days of gestation, whereas, those maternally disomic for Igf-II/H19 region (Mat.Dp.Dist. 7) have retarded growth and die at late gestation or immediately after birth (Sasaki et al., 1992
; Cattanach and Beechey, 1998
; Banerjee et al., 2000
).
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Finally, one might ask if there is any possibility of abnormal paternal chromosomes interfering with the development of healthy blastocysts. There are scenarios where this possibility could exist. As mentioned above, rapid cleavage and cell determination in preimplantation embryos are entirely reliant upon embryonic gene activation. The activation of housekeeping genes (polymerases, cell-cycle kinases and phosphatases, etc) and Oct-3/4 gene products, critical for maintaining pleuripotency of the embryo and determination of cell fate in blastocysts (Niwa et al., 2000), could occur from either of the two alleles. Maternal loss of function of any of these genes, resulting from high frequency of aneuploidy in human oocytes (Martini et al., 1997
), could be lethal in the absence of functional complementation by an activated paternal allele. Under such circumstances, inheritance of abnormal paternal chromosomal complements would lead to degeneration of 24-cell embryos. Genetic rescue of this type would however fail to prevent inheritance of defective parental alleles.
To summarize, blastocyst development reflects the macromolecular and enzymatic competence of the oocyte cytoplasm and is relatively independent of paternal genomic effect. The simplest way to overcome the possible adverse genetic consequences of abnormal paternal chromosomes in assisted reproduction, would be either minimizing sperm DNA damage or repairing damage prior to ICSI. This, however, is a daunting task because: (i) we do not know to what extent the structural genome (centromeric and non-centromeric heterochromatin) and protein coding genes are mutated in damaged spermatozoa; and (ii) the technology of repairing a damaged haploid genome is almost non-existent. The recent development of cytoplasmic transfer and oocyte fusion methods (Cohen et al., 1998; Tesarik et al., 2000
), raised the possibility of developing in-vitro sperm DNA repair systems and delivering the remodelled sperm nucleus to the oocytes.
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Notes |
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This debate was previously published on Webtrack, September 11, 2000
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References |
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