III. LA TRANSFORMACIÓN DEL SISTEMA DE ASENTAMIENTOS
2. Estructura urbana de los principales núcleos
In this chapter, I present a summary o f my results already published on the expression of the MPK gene in human preimplantation embryos and further extend these studies to later stage embryos. Previously, I have shown the presence of de novo transcripts of the paternally-inherited allele o f the M PK gene in embryos from the 1-cell to the 4-cell stages of preimplantation development. Here, using a RACE RT-PCR protocol, I have examined the expression of the M PK gene in embryos at later stages of development. However, although M PK transcripts are detected in embryos at the earlier 1-cell to 4-cell stages, no transcripts o f the M PK gene were detected in any of the nine 8-cell embryos analysed in this way. This seemed curious and warranted further examination.
A more sensitive RT-PCR approach was developed, reducing the sample preparation time to decrease mRNA degradation, and using a random hexamer- primed reverse transcription reaction. This approach was shown to be capable of detecting transcripts from both alleles of the M PK gene in a single fibroblast cell from an individual heterozygous at the MPK triplet repeat locus with a high efficiency (75 per cent). Using the RACE RT-PCR, transcripts of M PK were detected in single buccal cells (Daniels et al, 1995) and single fibroblast cells (data not shown) with an efficiency o f only -1 0 per cent. The analysis of HPRT transcripts in the same single cell or preimplantation embryo served as a positive control for the random primed reverse transcription procedures. Using this more sensitive RT-PCR approach, transcripts o f M PK were detected in preimplantation embryos at the 4-cell, 8-cell and
10-cell stages.
The difference between the detection of M PK transcripts using the two RT- PCR approaches seems significant, and if real requires explanation. Transcripts of M PK were readily detected in embryos from the 1-cell to the 4-cell stages using both approaches, whereas transcripts of M PK were only detected in embryos at the 8-cell stage using the random primed RT-PCR approach. In the mouse, maternal mRNA is degraded from the 1-cell to the 2-cell stage (Clegg and Piko, 1983) and maternal proteins or enzymes are degraded from the 8-cell stage (for further discussion see Harper and Monk, 1983). It is possible that an overall decrease in M PK mRNA (by degradation o f maternal mRNA) occurs from the 4-cell to the 8-cell stage of human preimplantation development, resulting in a lower level of M PK transcripts in 8-cell embryos detectable only when using the more sensitive, random hexamer-primed RT- PCR procedure. This might be observable in this study as a decrease in product intensity and/or efficiency in M PK transcript detection from the 4-cell to the 8-cell stages using the random primer RT-PCR procedure. However, I do not have sufficient data to speculate further at the current time. In order to demonstrate a real decrease in MPK transcription from the 4-cell to 8-cell preimplantation stages, quantitation o f the PCR products obtained using the random primed RT-PCR would have to be carried out.
Another possible explanation for the difference in the detection o f M PK transcripts using the two RT-PCR protocols may lie in the detection of only polyadenylated mRNA using the RACE RT-PCR protocol. Using the random primed RT-PCR approach, mRNA will be detected regardless of its polyadenylation status. The polyadenylation o f mRNA is one proccess by which a gene's expression may be
controlled, a polyadenylated status being associated with mRNA translation (see discussion, Chapter 10, 10.2.2.3; for review see Seydoux, 1996). Hence the ability of the RACE RT-PCR protocol to detect transcripts of the M PK gene in embryos at the 4-cell but not the 8-cell stage of development may reflect the de-adenylation and, therefore, inactivation o f M PK mRNA in human preimplantation embryos at the 8-cell stage.
The detection of de novo embryonic transcripts of the predominantly muscle- specific M PK gene at this early stage of human preimplantation development, described previously (Daniels et al, 1995) and studied in more detail in this chapter, is surprising. However, it is clear from these results and from the detection of de novo transcripts of the Y chromosome-linked genes, ZF7 and SPY (Ao et al, 1994; Fiddler et al, 1995), as early as the 1-cell stage of development, that the onset o f gene transcription from the zygotic genome in human preimplantation embryos occurs for some genes at the 1-cell stage.
Although the expression of the MPK, SPY and ZFY genes is predominantly tissue- or developmental stage-specific, transcripts of these genes have been detected in a wide range of adult and fetal tissues (Koopman et al, \9 9 \ ,Y\x et al, 1993; Clepet et al, 1993). It is difficult to imagine a specific function for these genes in the 1-cell human preimplantation embryo and it is possible that the M PK transcripts in early human embryos may be due to the detection of illegitimate or “leaky” transcription by the highly sensitive RT-PCR procedures. Illegitimate transcription has previously been described as one copy o f mRNA present in 500 to 1000 cells (Chelly et al,
1988), although an estimate of illegitimate transcription levels depends on the sensitivity of the procedures used. Here, transcripts of the MPK gene are detected in just a few cells. If this is our indication of the level of “illegitimate transcription” then we would have to conclude that all genes are expressed to some degree in some cells. However, this does not seem to be the case as is discussed in Chapter 4 and Chapter 10. Alternatively, the presence of these de novo M PK transcripts at this early stage of development may reflect a more specific derepression of gene transcription of certain genes in the preimplantation embryo, perhaps as a consequence o f some aspect of fertilisation. These questions are further addressed in Chapter 4.