A hypothesis on the origin of germ cell mutation and evolutionary role of extraembryonic mutation: Opinion

Jeffrey W. Persson

Sydney IVF, 4 O'Connell Street, Sydney, NSW, 2000, Australia


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 Introduction
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Intracytoplasmic sperm injection (ICSI) has provoked debate and interest in genetics (Persson et al., 1996Go). An interesting observation has been the finding of an excess of sex chromosome aneuploidy in ICSI offspring (Tournaye et al., 1997Go). It has been postulated that these errors are paternally derived. Interestingly, in the follow-up examination of eight ICSI pregnancies affected by sex chromosome aneuploidy the fathers were not found to have peripheral blood evidence of sex chromosome aneuploidy (M.Bonduelle, personal communication). A finding that would suggest confined germ cell 46,XY/47,XXY mosaicism in the fathers as a possible cause and these parents should be advised of an increased risk of recurrence. The normal prevalence of Klinefelter syndrome is 1 in 1000 live births (Nielsen and Wohlert, 1990Go). Recently performed molecular genotyping of the X-chromosomes of two boys with non-mosaic 47,XXY suggests that both sibs had inherited a paternal X-chromosome (Woods et al., 1997Go). It is plausible that confined germ cell 46,XY/47,XXY mosaicism in the father was the cause. The father had normal fertility and two further children with karyotypes 46,XY and 46,XX. It has been stated that gonadal mosaicism is most likely to be seen in those autosomal dominant or X-linked diseases where reproduction is disadvantaged (Strachen and Read, 1996Go).

Mosaicism is observed in 1–2% of placentas during first trimester chorionic villus sampling. These samples are derived from extraembryonic tissue, the trophoblast (Gardner and Sutherland, 1996Go). Different genetic cell lines might occur in the blastocyst through an early post-zygotic mutation (Figure 1Go). In the newly formed embryo the centrally placed, third or fourth generation blastomeres are displaced to the centre of the morula and become the inner cell mass. There is some exchange of blastomeres between the inner cell mass and the outer cell mass (Larsen, 1997Go). The outer cell mass makes up the greater proportion of the blastocyst volume (Fleming, 1987Go; Hardy et al., 1989Go). On day 6, the epiblast or primary ectoderm develops from the embryonic pole contiguous with the trophoblast/outer cell mass. It is plausible that an early mutation in a single blastomere leads to one pole of the embryoblast and its adjacent trophoblast possessing the same confined mutation. The affected pole of the epiblast would then subsequently develop to form part of the primitive streak region. It is from the primitive streak/epiblast that the primordial germ cells originate, detach and migrate to the yolk sac wall (Lawson and Hage, 1994Go). Both the haematopoietic stem cells and the primordial germ cells develop from the caudal primitive streak ectoderm. Thus if exchange of post-zygotic mutated blastomeres from the outer cell mass to inner cell mass occurred, one could reasonably assume that some of the cells which develop at the caudal end of the primitive streak/epiblast would similarly contain the mutation. A recent model of germline and trophectoderm separation has been proposed whereby differentiation begins as early as the 2-cell stage (Edwards and Beard, 1997Go).



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Figure 1. Proposed model of mosaic cell lineage in germline and trophoblast. ICM = inner cell mass.

 
Primordial germ cells migrate to a mass of extraembryonic mesoderm at the caudal end of the embryo and then to within the endoderm of the yolk sac wall. The haematopoietic stem cells develop similarly and are recognizable around the 17th day in the extraembryonic mesenchyme that surrounds the yolk sac endoderm. In the cytogenetic laboratory at Sydney IVF, an interesting experiment of nature was recently observed that lends support to the possibility that confined germ cell mosaicism is an error, post-zygotic or not, originating in the outer cell mass (Jahnke et al., 1997Go). A mosaic isochromosome trisomy 21, karyotype 46,XX,I(21)(q10)[13]/46,XX[2], was detected in a first trimester chorionic villus sample examined in three independent long-term cell cultures. The karyotype, at subsequent amniocentesis from 26 colonies in six in-situ cultures, was 46,XX. However, cells sampled from the amniotic cavity originate from the inner cell mass. Then, in the neonatal period, the same mosaicism was detected in the peripheral blood. Resolving these seemingly discrepant findings suggests that the isochromosome 21 cell line began as an error encompassing both the haematopoietic and germ cell progenitors of the caudal primitive streak. The placental cell line with the isochromosome was confirmed in the placenta postnatally using a fluorescent in-situ hybridization probe.

The cell line with the isochromosome was found in four out of 100 cells examined from umbilical cord blood. Subsequent physical examinations of the infant, up to 12 months of age, have revealed no stigmata of trisomy 21. At 6 months of extrauterine life, the population of peripheral blood cells that contained the isochromosome 21 mutation was two cells in 200. The relative decline, at 6 months, in the fraction of blood cells containing the translocation might be explained by selection through decreased survival of the mutated cell line. The cells, alternatively, might represent the fraction of fetal lymphocytes still surviving and of extraembryonic, yolk sac origin. There are similar examples from the literature of mitoses, prior to the separation of the germline, contributing to both the germline and haematopoietic stem cells (Gardner et al., 1994Go; Gregory et al., 1997Go; Sciorra et al., 1992Go).

It is interesting to speculate on the possibility of mosaicism being present in the ovarian germ cells, in this patient, as the germ cells also originate from the caudal primitive streak. The presence of germ line mosaicism, despite the absence of evidence for mosaicism in the peripheral blood of the fathers, might account for ICSI offspring affected by sex chromosome aneuploidy. As a corollary, the offspring of the mosaic pregnancies should receive appropriate genetic counselling concerning the possibility of subfertility and recurrent miscarriage. The placental function of the genetically abnormal placenta is likely to be a mechanism determining the survival of such experiments of nature (Pittalis et al., 1994Go).

There has been speculation on the origin of Y chromosome mutations associated with spermatogenic defects (Edwards and Bishop, 1997Go). The possibility of Y-deletion mosaicism arising in germ cells has been suggested (Kent-First et al., 1996aGo,bGo; Reijo et al., 1996Go). The observers noted no deletions in the peripheral blood of several fathers of Y-deletion-affected offspring. Consequently one might assume that the Y chromosome mutations arise similarly in the extra-embryonic cell mass before primordial germ cell migration into the embryo. The only way to test these theories is to obtain tissue from the paternal testes and perform deletion studies and cytogenetics. Edwards and Bishop (1997) suggested that the conundrum might be examined by analysis of a single spermatozoon. This is likely to be exceedingly difficult, as it would involve distinguishing between the failure of a single cell reaction to amplify under polymerase chain reaction (PCR) (allele dropout) and the occurrence of a true deletion. This is the same technical problem that presently bedevils attempts at PCR-based preimplantation genetic testing. Cytogenetic studies for the sex chromosome could be a more robust way of looking for paternal germ cell mosaicism, although the accuracy of fluorescent in-situ hybridization (FISH) for detection of deletions in individual spermatozoa remains to be proven.

It has been proposed that natural selection depends on the genetic variability generated by mutations arising in the germ cells of the testis (Short, 1997Go). However, a facility for genetic experiments in the extraembryonic cell mass might have the evolutionary advantage of allowing mutations in germ cells without a deleterious change of genetic constitution in the future soma of the inner cell mass. Any potentially deleterious mutations would then be confined to the germ cell line and thus be secluded from the host soma. Furthermore, the occurrence of a mosaic germ line might explain instances of recurrent miscarriage and subfertility. From an evolutionary perspective this hypothesis suggests an important mechanism allowing natural selection through genetic diversity in the subsequent generation of embryos. It might also explain how mosaicism confined to the germ cell line is associated with aneuploidy, and deletions, in the embryos of males affected with infertility.


    References
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Edwards, R.G. and Beard H. K. (1997) Oocyte polarity and cell determination in early mammalian embryos. Mol. Hum. Reprod., 3, 863–905.[Abstract]

Edwards, R.G. and Bishop, C.E. (1997) On the origin and frequency of Y chromosome deletions responsible for severe male infertility. Mol. Hum. Reprod., 3, 549–554.[Abstract]

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Hardy, K., Handyside, A.H. and Winston, R.M. (1989) The human blastocyst: cell number, death and allocation during late preimplantation development in vitro. Development, 107, 597–604.[Abstract]

Jahnke, A., Wright, D., Boogert, T. and Roberts, C. (1997) Diagnosis of mosaic isochromosome trisomy 21 confined to the placenta. Appl. Cytogenet., 23, 80.

Kent-First, M.G., Kol, S., Muallem, A. et al. (1996a) Infertility in intracytoplasmic-sperm-injection-derived sons. [Letter.] Lancet, 348, 332.

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