Las infraestructuras como elemento básico de integración territorial

In the mouse, both X chromosomes are active in female preimplantation embryos (Kratzer and Gartler, 1978; Epstein et al, 1978; Monk and Harper, 1978). Inactivation of the paternal X chromosome occurs first in the extra-embryonic trophoblastic cells of the developing blastocyst (Takagi and Sasaki, 1975), followed by random inactivation of either the maternal or paternal X chromosome in the cells which form the embryo proper. This random X-inactivation is maintained in the somatic cell lineages throughout the lifetime of the individual. However, in female germ cells, the inactivated X chromosome is reactivated during oogenesis (Kratzer and Chapman, 1981). In males, the single X chromosome is active throughout all pre- and postnatal life, except during spermatogenesis, where it is inactivated at, or just prior to, the onset of meiosis (see Salido et al, 1992; Richler et al, 1992). This

inactive X chromosome in sperm is reactivated shortly after fertilisation.

In the human, less is known about the timing o f X-inactivation in early development due to the scarcity of material available for research purposes. However, recently, experiments have shown that preferential paternal X-inactivation has occurred in first trimester human trophoblastic cells (Goto et a l, 1997). Previously, preferential expression of the maternal allele of G6PD (glucose-6-phosphate dehydrogenase) and, hence, paternal X-inactivation, has been demonstrated in term placentae at birth (Ropers et al, 1978; Harrison, 1989). However, conflicting results have been obtained showing expression of both maternal and paternal alleles of the G6PD gene in chorionic villi cells

from both fetal and newborn placenta (Migeon and Do, 1979; Migeon et al, 1985). In the chorionic villus samples this difference in results may be explained by the difficulties encountered when isolating the extra-embryonic trophoblastic cells from the from the mesodermal cells. The presence of any mesodermal cells in the trophoblast samples being studied would result in the detection of expression of both maternal and paternal G6PD alleles. In addition, the long term culture of the chorionic villi cells used by Migeon et al (1985) may have resulted in cultures enriched for mesoderm cells and/or the characteristics of fresh trophectoderm cells may have been altered in culture. It appears likely, therefore that the paternal X chromosome is preferentially inactivated in the extra- embryonic cell lineages of the developing human embryo, as is the case in the mouse.

In human spermatogenesis, the expression of the X IST gene and the presence o f structures characteristic of an inactive X-chromosome in silver stained human pachytene spermatocytes (see Salido et al, 1992; Richler et al, 1992) indicate that X- inactivation occurs, as in mouse spermatogenesis. In addition, both X chromosomes have been shown to be active during human oogenesis (Gartler and Andina, 1976).

In this chapter, I describe initial data on the analysis o f X chromosome activity in human preimplantation development. The mRNA levels o f the X-linked gene, HPRT, in comparison to that of the autosomal gene, GAPDH, in female and male preimplantation embryos provides evidence as to the activity o f the two X chromosomes in female embryos compared to the single X chromosome in male embryos. An HPRTIGAPDH ratio in female:male embryos o f 2:1 indicates both X

chromosomes are active in the female preimplantation embryo. I have analysed HPRT and GAPDH transcripts in male and female embryos at the 8-cell stage, previously sexed and analysed for XIST expression. The embryonic complement o f cDNA in the first round PCR reaction mix, following the amplification of a specific cDNA, enables further information to be obtained from the rare human preimplantation embryo samples. The ratio of HPRTIGAPDH in the two female preimplantation embryos analysed was found to be almost twice that in the two male preimplantation embryos analysed. However, the low number of embryos available for analysis prevents any statistical significance being attached to these results, and ideally this work should be continued with fi-esh samples. Nevertheless, this data is strong preliminary evidence of two active X chromosomes in human female preimplantation embryos. The simultaneous analysis of HPRT and GAPDH expression in all four embryos in the same experiment reduces the possibility of PCR reaction variation. Although it is possible that variations in the efficiency of PCR amplification could occur from sample to sample, it is unlikely that this would have resulted in such a distinct difference between the ratios of HPRTIGAPDH cDNA seen between the female and male embryos.

An alternative RT-PCR approach to determine whether both X chromosomes are active in human female preimplantation embryos would be to amplify a polymorphic region of an X-linked gene expressed in preimplantation embryos. In female embryos heterozygous for the polymorphism, transcripts from each X chromosome could be identified and thus, the status of activity o f the two X

chromosomes could be deduced. However, due to the requirement o f heterozygous female embryos, this approach would also be limited by the low number of embryos available for research. The analysis of a gene with a polymorphic site showing a high frequency o f heterozygosity, e.g. the androgen receptor gene (Busque et al, 1994), would reduce this problem. However, it is not known whether this gene is expressed at this stage of human development.

Previously, the ratio of hprt/aprt enzyme activity in mouse male and female embryos has shown that both X chromosomes in the mouse female preimplantation embryo are active until the blastocyst stage when the paternal X chromosome is inactivated in the extra-embryonic cell lineages (Monk and Harper, 1979). Here, I provide evidence for the activity of both X chromosomes in female human preimplantation embryos at the 8-cell stage. It appears therefore, that the activity of the X chromosomes in female human preimplantation embryos follows a similar pattern to that in the mouse, with both X chromosomes active in the early preimplantation embryo, followed by the preferential paternal X-inactivation in the extra-embryonic cell lineages (Monk and Harper, 1979; Harrison, 1989; Goto et al, 1997). The timing of X-inactivation in the human embryo proper is still not known. The similarities in the pattern of X chromosome inactivation in female embryos in both mouse and human supports the idea that a similar mechanism is responsible for X-inactivation in both species.