Molecular Human Reproduction, Vol. 7, No. 2, 147-154,
February 2001
© 2001 European Society of Human Reproduction and Embryology
Embryology |
Quantitative measurement of transcript levels throughout human preimplantation development: analysis of hypoxanthine phosphoribosyl transferase
1 Department of Reproductive Sciences and Medicine, Division of Obstetrics and Gynaecology, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, 2 School of Biology, University of Leeds, Leeds LS2 9JT, UK, 3 Centre Génétique, U393 Institut Necker, Hôpital des Enfants Malades, Paris, France and 4 Department of Obstetrics, Gynaecology and Human Genetics, Royal Victoria Hospital, McGill University, Montreal, QC H3A 1A1, Canada
Abstract
We have developed a competitive reverse transcription-polymerase chain reaction (RT–PCR) sensitive enough to detect and quantify as little as 2-fold differences in gene expression in individual oocytes and embryos throughout human preimplantation development. This RT–PCR assay can be tailored for the examination of any specific gene and so will give a unique insight into human preimplantation development. This technique was used to quantify the level of hypoxanthine phosphoribosyl transferase (HPRT) expression during preimplantation development and to correlate this with embryo sex. The amount of HPRT transcripts present in the unfertilized oocyte was equivalent to 7.7 fg of competitor cDNA. At the 4-cell stage there is a significant drop (P = 0.0006) to ~1.2 fg. There was no detectable difference in the HPRT levels between female and male embryos following 2 days of in-vitro culture. In contrast HPRT gene expression was higher in day 3 female embryos than in males. This is the first study to quantify gene transcripts throughout each stage of human preimplantation development and it indicates that the accumulated HPRT transcripts present in the unfertilized human oocyte undergo extensive destruction following fertilization. This work also suggests that X-inactivation occurs beyond the 8-cell stage of human preimplantation development.
HPRT/human preimplantation embryo/oocyte/quantification/RT-PCR
Introduction
Global embryonic gene activation occurs at the 4-8-cell stage in the human (Braude et al., 1988
; Tesarik et al., 1988). Prior to this, the initial stages of preimplantation development are dependent on the proteins and transcripts accumulated in the oocyte during oogenesis. This is demonstrated by the human embryo's ability to undergo cell cleavage to the 4-cell stage in the presence of the transcription inhibitor 
-amanitin (Braude et al., 1988).
The time at which the embryonic genome becomes active is characterized by both the expression of embryonic genes and the degradation of the maternal transcripts stored in the oocyte (Telford et al., 1990). Most of our current understanding of the amount and rate of degradation of oocyte-derived transcripts comes from work carried out in the mouse. Oocytes have been labelled in vitro with tritiated uridine to measure the proportions of rRNA and poly (A)+ content at different stages of oocyte maturation. In the fully grown mouse oocyte, the total amount of maternal mRNA is estimated at 83 pg/oocyte (De Leon et al., 1983). Between fertilization and the onset of embryonic gene activation at the late 2-cell stage, 70% of the total poly (A) RNA is lost (Bachvarova and De Leon, 1980; Clegg and Piko, 1983). Because this loss occurs prior to activation of the embryonic genome it must reflect the elimination of the maternal RNA stored in the oocyte.
To further characterize the changes in RNA content during mouse oocyte maturation, Northern blot analysis has been used to quantify specific gene transcripts during this period, and mRNA degradation has been demonstrated for tissue-type plasminogen activator (t-PA), actin, tubulin and hypoxanthine phosphoribosyl transferase (HPRT) (Huarte et al., 1987; Paynton et al., 1988). Although scientifically invaluable, this approach typically requires hundreds of oocytes and embryos to obtain sufficient quantities of RNA for each experiment and therefore cannot be used with human oocytes and preimplantation embryos as the numbers available for research are limited.
Although reverse transcription polymerase chain reaction (RT–PCR) has been widely used to detect the presence or absence of specific gene transcripts in individual human embryos (Pergament and Fiddler, 1998), information regarding the quantities of specific gene transcripts is extremely limited. This is because quantitative RT–PCR is technically challenging due to the high levels of amplification required for embryos and therefore this approach demands stringent controls for variations in amplification efficiencies. In the absence of these controls, semi-quantitative RT–PCR can produce information on the relative changes of specific gene transcripts, but this method fails to provide quantitative data. Semi-quantitative RT–PCR has been used to characterize the patterns of expression for cell cycle genes c-mos and cyclin B1 and ß-actin mRNA (Heikinheimo et al., 1995); however, it would be of added value to know the actual amounts of transcripts present. Quantitative RT–PCR has been used to determine the different amounts of ß-actin and interleukin-1 receptor type I (IL-1R tI) mRNA in individual blastomeres from tripronucleate human embryos around the 8-cell stage (Krussel et al., 1998). These blastomeres produced large variations in the mRNA content between cells from the same embryo.
The main aim of this study was to develop a quantitative assay sensitive enough to examine the expression of specific mRNA transcripts throughout the different stages of human preimplantation development, including the critical period at which global embryonic gene activation occurs. Such an assay will enable us to quantify the expression of candidate genes that play a key role in preimplantation development. This will further our understanding of preimplantation development and will also provide a means of identifying the most viable embryos for IVF-embryo transfer.
Initially we have chosen to examine the changes in HPRT specific gene expression in normally fertilized embryos throughout human preimplantation development. HPRT is an X-linked house-keeping gene, required for purine salvage. A deficiency in the enzyme results in the human sex-linked disease Lesch-Nyhan syndrome, which is characterized by mental retardation and self-mutilation. The HPRT gene is constitutively expressed in all cells and tissues. The purines hypoxanthine and adenosine are components of follicular fluid (Eppig et al., 1985) and the purine metabolic pathways are critical participants in the regulation of meiotic arrest in the mouse (Downs et al., 1985). As a typical house-keeping gene, its pattern of expression may represent that of a number of other genes, which makes HPRT an ideal gene for this study. The examination of X-linked HPRT gene expression is of additional interest as it can provide information on the timing of X inactivation during human preimplantation development, which is currently unknown.
Materials and methods
Human oocytes and embryos
Women underwent ovulation induction and oocytes were collected by transvaginal ultrasound-guided aspiration and inseminated with prepared spermatozoa (day 0) as previously described (Rutherford et al., 1988). Oocytes were examined the following morning, 19–20 h post insemination, and classified as normally fertilized or polyspermic depending upon the presence of two or more pronuclei respectively. Fertilized embryos were cultured in Earle's balanced salts solution (EBSS) supplemented with 10% heat-inactivated maternal serum (Hardy et al., 1989). Oocytes which failed to be fertilized and surplus embryos following transfer were used with the couples' informed consent. Embryos used for each stage of development were matched for both embryo age and grade. Embryos were graded (Dawson et al., 1987) and embryos of grade III or less were not included in the study. Both unfertilized oocytes and pronucleate stage zygotes were collected at the same time on day 1, ~24 h post insemination. This work was carried out under a Human Fertilisation and Embryology Authority licence, with local approval from the Research Ethics Committee of the Imperial College School of Medicine.
Reverse transcription
After removing the zona pellucida with acidified Tyrode's solution (pH 2.4) and washing in phosphate-buffered saline (PBS, Gibco), individual oocytes and embryos were lysed in 5 µl of lysis buffer [0.5% NP-40, 10 mmol/l Tris (pH 8.0), 10 mmol/l NaCl and 3 mmol/l MgCl2] on ice (Gilliland et al., 1990). The lysate was then made up to 20 µl with reverse transcriptase (RT) buffer (50 mmol/l Tris–HCl (pH 8.3), 75 mmol/l KCl, 3 mmol/l MgCl2, 10 mmol/l dithiothreitol (Pharmacia), 300 pmol oligo (dT)12–18 primer (Pharmacia) 0.5 µmol/l dNTP (Pharmacia) and 100 units of M-MLV reverse transcriptase and incubated at 37°C for 1 h. The reverse transcriptase enzyme was omitted for the negative controls, which were included in each experiment. The reaction was terminated by heating at 95°C for 5 min. The tubes were spun for 1–2 min and the supernatant used for nested PCR. In some cases, reverse transcription was not carried out immediately, in which case lysates were snap-frozen in liquid nitrogen and stored at –70°C. RT–PCR products were verified by sequencing analysis (data not shown).
Polymerase chain reaction
Following reverse transcription, 20 µl of the cDNA product was made up to 30 µl with PCR buffer [10 mmol/l Tris–HCl, pH 8.3, 50µmol/l of each dNTP, 2.5 units Taq polymerase (AmpliTaq, Perkin Elmer), 0.4 µmol/l of each primer], covered with 50 µl of silicone oil and denatured at 94°C for 2 min. Cycling conditions were the same for all reactions: denaturation for 45 s at 94°C, annealing at appropriate temperature (Table I) for 60 s and extension at 72°C for 90 s for 30 cycles. Each PCR was completed with a final extension step at 72°C for 5 min. A nested PCR protocol was used only to generate the competitor template, all other PCR reactions for the quantitation used a single primer pair. For the nested reaction, 2 µl of the first amplification product was added to freshly prepared PCR mix as above. Primer positions are shown in Figure 1A, and expected sizes for each amplified product are given in Table I.
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Restriction digestion of PCR products
To distinguish between competitor and target (embryonic) HPRT amplification products, 30 µl of amplification product was incubated overnight with 5U of Hpa II restriction enzyme and its reaction buffer at 37°C as recommended by the manufacturers guidelines.
Southern transfer, hybridization and densitomery analysis
The digested 30 µl PCR products were separated on a 3% NuSieve agarose gel, and then blotted onto Hybond N membrane (Amersham, UK) (Sambrook et al., 1989). Nucleic acids were cross-linked to the membrane using the Biorad Gene Linker UV chamber (Biorad). The cDNA probe was radiolabelled by incorporation of [32P]dCTP, carried out according to manufacturer's instructions using a Nick translation kit (Boehringer Mannheim). Prehybridization of the nylon membrane was carried out for at least 5 h prior to addition of the radiolabelled probe. An oligo internal to the digested 225 bp PCR fragment was used as the probe for the Southern blot. Hybridization of the radiolabelled probe was carried out overnight at 42°C. The membrane was then washed twice in 2x standard saline citrate (SSC)/0.1% sodium dodecyl sulphate for 30 min each at 42°C. The membrane was finally exposed to X-ray film (Kodak XAR-2) overnight to obtain an autoradiographic image, which were scanned using an Agfa Studio II Si System scanner and densitometry analysis was carried out using Scan Analysis software (Biosoft®, Cambridge).
Fluorescence in-situ hybridization (FISH) on single blastomeres
Individual blastomeres were fixed to slides according to a published method (Harper et al., 1994). Prior to hybridization, slides were initially incubated in 0.1 mg/ml of pepsin for 20 min at 37°C. After washing in PBS, the slides were incubated in 1% paraformaldehyde for 10 min at 4°C. Slides were washed again in PBS and then distilled water before dehydrating by incubation for 5 min in 70%, 90% and then 100% ethanol at room temperature. Hybridization of the fluorescent probe was carried out according to a modified version of manufacturer's recommendations using the Triple colour DNA mixture (VysisTM, Surrey, UK). Slides were placed on a hot block at 73°C for 5 min to co-denature both the nuclear and probe DNA. Slides were then placed in a dark humidified container at 37°C for 45 min to allow hybridization of the probe to nuclear DNA under a cover slip. After hybridization, cover slips were removed from the slides, which were then washed in 0.4xSSC/0.3% NP-40 at 73°C for 2 min. The slides were then washed at room temperature in 2xSSC/0.1% NP-40 for 1 min and left to air dry. Slides were mounted with antifade Vectarshield (Vectar Laboratories, Burlingame, CA, USA) containing 1.2 µg of DAPI which counterstains the nucleus. A cover slip was placed over the target area of each slide and air bubbles were removed by applying pressure to the cover slip with a pair of blunt forceps. Slides were either scored immediately on a Axophot fluorescent microscope (Zeiss), with a CCD camera controlled by SmartCaptureTM software (Vysis), or stored for up to 3 months in the dark at –20°C.
Statistical analysis
Statistical analysis was performed using an equal variance Student's t-test with P < 0.05 considered significant.
Results
Strategy for quantitative RT–PCR
The assay is based on the use of a reference standard, referred to as the competitor, which is co-amplified in the same reaction as the sequence of interest (Becker-Andre and Hahlbrock, 1989). The standard is a synthetic template, rather than an endogenous gene, which is identical to the target sequence, except for a single base pair mismatch which introduces a novel Hpa II site in the competitor template. A fixed amount of competitor cDNA is added to different samples and the mixture is subjected to single step PCR. Both the standard and the target sequence are amplified using the same primers, producing PCR products of identical size, therefore allowing the competitor to control for any tube-to-tube variation in amplification efficiency. The competitor template is then distinguished from the embryonic product following restriction digestion with Hpa II which results in the competitor being 25 bp smaller than the 225 bp embryonic product. The initial amount of cDNA reverse-transcribed from the embryo sample can be determined by comparing amplification from the embryonic cDNA with that of the known amount of competitor template added to the PCR reaction. Results are expressed as a percentage of the competitor band.
HPRT primer selection and construction of the competitor template
Figure 1A
demonstrates how HPRT outer primer pair OP1 and OP2 and nested primer pair DP and NP2 were designed to amplify cDNA reverse-transcribed from pooled blastocysts to produce the competitor template. Primer details are given in Table I. Figure 1B shows how primer DP introduces a novel Hpa II restriction site when used to amplify the cDNA to accumulate the competitor template. To derive absolute quantitative information by competitive PCR, the principal condition that must be fulfilled is that the target and competitor sequences are amplified with equal efficiencies using identical primer pairs. Therefore NP1 and NP2 were used in subsequent experiments to amplify both the target and competitor cDNA without affecting the Hpa II restriction site in the competitor cDNA. The RT–PCR product was visualized on an agarose gel and the cDNA band was cut out and extracted from the gel according to manufacturer's instructions (Wizard DNA purification system, Promega, Southampton, UK). The cleaned product was then quantified by spectrophotometry and used as the competitor control template in subsequent experiments.
Preliminary PCR
Because the amount of product that accumulates at the plateau phase of the PCR reaction does not depend on the amount of starting material, it is necessary to quantify the PCR products while the PCR reaction is still in the exponential phase. To characterize the kinetics of the HPRT PCR, a cycle profile was carried out using 20 fg of cDNA. PCR products were visualized by Southern hybridization. A plot of cycle number against optical density of scan area shows that the PCR begins to plateau at ~23 cycles (data not shown). As a consequence of these results, 19 cycles were used in subsequent competitive PCR analyses. Radiolabelled probes were used as standard, to increase the sensitivity of the assay while remaining in the exponential phase of the PCR reaction. To obtain accurate quantification, the embryonic target and competitor sequence must be present in similar amounts. To achieve this, a concentration profile of competitor template was run alongside a single embryo, using 19 cycles. The HPRT gene transcripts from the single embryo showed comparable amplification to 10 fg of competitor cDNA (data not shown) and this quantity of competitor was used in subsequent competitive PCR analyses.
Assessing the sensitivity of the quantitative RT–PCR
To test the sensitivity of this approach, single and pooled unfertilized oocytes were collected and the levels of HPRT mRNA in different samples were examined (Figure 2). This experiment was performed on groups of 1, 2 and 4 unfertilized oocytes and repeated on at least two separate occasions. Comparison of the top unfertilized oocyte-derived bands with the lower competitor bands in Figure 2A allows the level of HPRT mRNA to be assessed for different numbers of unfertilized oocytes. Figure 2B gives a comparison of cDNA PCR amplification from the competitor only and embryo only to demonstrate that the amplification from each band is comparable, and that the competitor cuts to completion. In each experiment a single embryo was processed without reverse transcriptase to act as a negative control. Figure 2C shows the increase in HPRT mRNA level with increasing oocyte number, expressed as a percentage of the competitor band. This graph demonstrates that the quantitative RT–PCR developed here is sensitive enough to detect as little as 2-fold differences between different samples. The fact that there are not precise doublings between samples may reflect the natural variation in the amount of HPRT mRNA present in individual oocytes as well as the small numbers of samples used.
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HPRT stage specific gene expression
Figure 3
shows a representative selection of the embryos used for each stage of development which were matched for both embryo age and grade. Figure 4A is an autoradiograph from a Southern blot demonstrating the changes in HPRT gene expression in single human oocytes and embryos at defined stages of preimplantation development. This experiment was performed on three separate occasions and gives the results from 34 oocytes and embryos. The level of HPRT mRNA detected at each stage of development can be calculated by relating the optical scan density of the top embryonic band with that of the lower competitor band. Because the competitor template is co-amplified with the embryonic cDNA, and because the amount of competitor added to the initial PCR reaction is known (10 fg), we can use this to determine the amount of HPRT mRNA present at each stage of preimplantation development studied. Figure 4B is a plot of the data from between five to eight individual oocytes and embryos, analysed from four repeat experiments. There is equivalent to 7.7 fg or 31 000 molecules of HPRT competitor cDNA at the unfertilized oocyte stage, which is a high level in comparison with the other stages of development. This high level is maintained at a level of 7 fg at the 1-cell pronucleate stage, following fertilization. Both tripronucleate and single pronucleate zygotes were included in the study, as comparisons of the level of HPRT mRNA detected for each sample showed no significant difference (data not shown). Four-cell embryos were collected after a further 24 h in culture, 2 days post insemination. Figure 4 shows an 82% reduction in the level of HPRT mRNA to a level equivalent to 1.2 fg, between the 1-cell pronucleate stage and the 4-cell stage of preimplantation development. This striking drop between days 1 and 2 post insemination is statistically significant (P = 0.0006). The levels of HPRT mRNA remained low at the 8-cell (1.8 fg) and blastocyst (1.5 fg) stages of embryo development.
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Stability of HPRT gene transcripts
To assess the stability of HPRT mRNA during human preimplantation development, and to further investigate the stimulus for the dramatic degradation of the transcripts between days 1 and 2 post insemination, unfertilized oocytes from a single treatment cycle were collected on different days post insemination. Figure 5A
is an autoradiograph demonstrating the level of HPRT mRNA in eight unfertilized oocytes collected on days 1, 2, 3 and 6 of successive in-vitro culture. Figure 5B is a plot of the relative HPRT mRNA levels on days 1 to 6. This graph demonstrates that unlike the stage specific profile, the amount of HPRT mRNA remains constant right through to day 6 of in-vitro culture, with no dramatic drop between days 2 and 3. The persistence of HPRT transcripts up to 6 days of in-vitro culture shows the remarkable stability of these maternally derived transcripts, assuming the oocytes are transcriptionally inactive.
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HPRT gene expression in sexed human preimplantation embryos
Cleavage stage embryo biopsy was carried out on 13 individual embryos at either the 4-cell stage on day 2 of in-vitro development, or at the 8-cell stage on day 3 of in-vitro culture. During the biopsy procedure a hole was made in the embryo's outer coat, the zona pellucida, and a single blastomere was carefully removed from each embryo by creating negative pressure in a fine aspiration pipette. Use of this biopsy procedure meant that individual blastomeres could be removed from each embryo without causing any damage to the remaining cells. Because embryos were matched for cell number prior to embryo biopsy, as well as stage of development, the removal of a single cell from each embryo meant that embryo sizes were consistent on each day studied. The biopsied cell was then fixed on a slide for sex determination by FISH analysis. Figure 6A
demonstrates how the sex of individual embryos was identified using FISH following the removal of a single blastomere from 4-cell (day 2) or 8-cell (day 3) embryos. Males were identified by the presence of two aqua signals, indicating the presence of two chromosomes 18, a single green signal and a single red signal, indicating one chromosome X and one chromosome Y respectively (Figure 6Ai). Female embryos were identified by the presence of two aqua signals, indicating the presence of two chromosomes 18, and two green signals indicating two X chromosomes (Figure 6Aii). Individual embryos were analysed by quantitative RT–PCR and the results pooled according to embryo sex and the stage of development. Figure 6B is an autoradiograph demonstrating the changes in HPRT gene expression in single sexed human embryos 2 and 3 days post insemination. Figure 6C illustrates the quantitative analysis of HPRT mRNA in these embryos. On day 2 post insemination, there was no difference in the level of HPRT mRNA between male and female embryos, but on day 3 post insemination, the HPRT gene expression in male embryos was lower than in females.
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Discussion
We have developed a quantitative, competitive RT–PCR sensitive enough to measure mRNA levels in single human oocytes and embryos. Using this technique, the expression pattern of HPRT mRNA has been characterized throughout human preimplantation development. Previously nothing was known about the amount of specific mRNA stored in the human oocyte and how quickly these transcripts are lost following ovulation. This study shows that there is a high level of HPRT transcripts, equivalent to 7.7 fg of competitor cDNA on day 1 of in-vitro culture, and this was rapidly reduced to 1.2 fg by day 2. This significant loss must reflect the loss of the maternal transcripts inherited from the oocyte as this process is complete by the time the embryo has reached the 4-cell stage, when expression from the embryonic genome is initiated (Braude et al., 1988; Tesarik et al., 1988). Because the loss of HPRT transcripts only occurs following fertilization and subsequent embryo cleavage, one of these events is probably the trigger which directs the dramatic degradation of this stable message. Degradation presumably results from the act of fertilization itself, although the underlying mechanism is largely unknown. It has been suggested that de-adenylation of mRNA may account for the observed decline, as an oligo (dT)-based reverse transcription method was used. However, the oligo (dT) approach works well on all mRNA since de-adenylation does not remove all of the poly (A) tail, leaving ~50 nucleotides in the case of mouse HPRT (Paynton and Bachvarova, 1994). As the oligo (dT) method will therefore transcribe adenylated and de-adenylated alike, de-adenylation cannot be an explanation for the drop in HPRT transcripts detected.
A dramatic fall in HPRT mRNA levels also occurs during mouse preimplantation development as detected by Northern blot analysis (Paynton et al., 1988). This work reported a 77% drop between the ovulated oocyte stage and the late 2-cell stage, when global activation of the mouse embryonic genome occurs. As this drop has also been demonstrated in other mammalian systems (Telford et al., 1990), it appears to be characteristic of normal mammalian preimplantation development, and may reflect the successful switch from maternal to embryonic gene expression. If this is the case, the degradation of oocyte-derived maternal transcripts may be indicative of an embryo's developmental potential.
It is clear that the level of accumulated gene transcripts present in the oocyte is significantly higher than that required by the embryo for its normal development to the blastocyst stage. Prior to ovulation, the oocyte has accumulated a mass of RNA and proteins to support early embryonic development. Any loss of maternal transcripts due to their instability may compromise the ability of the oocyte to support the initial development of the embryo, prior to the onset of embryonic gene expression. A combination of the high stability of oocyte-derived transcripts and a transcript level in excess of that required for normal preimplantation development, may ensure the developmental potential of the oocyte is not compromised, even over a substantial period of time.
Our results show no increase in HPRT gene expression following activation of the embryonic genome at the 4- to 8-cell stage, or even during development to the blastocyst stage on day 5 post insemination. This is perhaps surprising considering the significant increase in cell numbers at the blastocyst stage as well as the full activity of the embryonic gene at this later stage of preimplantation development. However, it may be relevant that there is no significant increase in cell mass during this developmental period (Figure 3).
As the quantitative RT–PCR used in this study is sensitive enough to detect as little as 2-fold differences in gene expression (Figure 2C) we have been able to examine the level of HPRT gene expression in relation to embryo sex. There was no detectable difference in the level of HPRT mRNA between male and female embryos following 2 days of in-vitro culture, and indeed the overall level of expression was above that found for the stage specific expression study (compare Figures 4 and 6). This may simply be due to the fact that the sexed embryos were processed earlier on day 2 and the higher levels reflect the fact that the degradation of the maternal, oocyte coded mRNA was incomplete. On day 3 of in-vitro culture HPRT gene expression in male embryos appears to be less than that of females. The difference in HPRT transcript level between male and female embryos is not 2-fold; this may reflect the persistence of a small proportion of maternal transcripts below the level of embryo coded message on day 3 of preimplantation development. The observation that female embryos express higher levels of HPRT mRNA than male embryos suggests that X-inactivation has not yet occurred and the female still possesses two active X-chromosomes on day 3 of in-vitro development. This situation is comparable with the mouse, as the female embryo still possesses two active X-chromosomes during the initial cleavage stages and therefore produce twice as much X-linked gene product as the males (Kratzer and Gartler, 1978).
A deficiency in the enzyme HPRT results in Lesch-Nyhan syndrome, which is characterized by compulsive self-injurious behaviour and early death. For PGD of single gene defects, PCR has been used to amplify two target DNA molecules from single cells biopsied from cleavage stage embryos (Handyside et al., 1989, 1992) and this approach has been used for the diagnosis of Lesch-Nyhan syndrome in embryos from a couple at risk of transmitting the disease to their offspring (Ray et al., 1999). Recently, because mRNA is more abundant, RT–PCR has been used to amplify transcripts from single cells biopsied from embryos at around the 8-cell stage in a couple at risk of Marfan syndrome, which is an autosomal dominant condition caused by defects in the FBN1 gene (Eldadah et al., 1995). However, use of RT–PCR as a diagnostic tool for PGD requires careful evaluation as the use of coding sequence polymorphisms has shown that RT–PCR can fail to detect a significant proportion of embryonic transcripts during human preimplantation development and that this is most probably due to the persistence of oocyte-derived transcripts (Taylor et al., 1997). Hence, knowledge of the timing of the loss of these oocyte transcripts is essential for those considering using RT–PCR for PGD. The work presented here suggests that most oocyte-derived transcripts are lost on day 2 of in-vitro development at the 4-cell stage. The lack of increase in HPRT mRNA level and the lack of detectable differences between female and male embryos on day 2 of human preimplantation development suggest that expression from the embryonic gene is low and may still be masked by residual oocyte-derived transcripts until later stages of development.
Differences in HPRT gene expression were detectable between male and female embryos on day 3 of in-vitro development. Although the number of embryos analysed was too small for the results to be significant, this suggests that the embryonic genome is active and the embryonic transcript level is above that of any residual oocyte coded message at this stage. Consequently RT–PCR is probably best considered for PGD following cleavage stage embryo biopsy on day 3 or blastocyst stage embryo biopsy on day 5 or 6 of in-vitro development, although the level at which the oocyte-derived maternal transcripts persist at these later stages of preimplantation development must be determined more exactly for each gene of interest.
Examination of both global and specific gene expression enables us to investigate the process of normal human preimplantation development. This is also of clinical significance, because of the possibility of identifying more viable embryos for IVF-embryo transfer, as well as the use of RT–PCR for specific diagnosis for PGD. Real time RT–PCR technology has also been used to analyse gene expression in human oocytes (Steuerwald et al., 2000). This method has the advantage of providing rapid and easily quantifiable results. The method described in this paper, however, does not require the equipment necessary for real time RT–PCR and has the advantage that the target and control templates are present in the same tube. To our knowledge this work is the first study to characterize HPRT gene expression at the RNA level in individual human oocytes and embryos in relation to the stage of preimplantation development as well as the embryo sex. The massive degradation of maternal transcripts demonstrated in this work, which is most probably linked to fertilization and activation of the embryonic genome, may serve as a marker for embryo viability. This work provides data not only of clinical significance but of scientific value, offering a unique insight into some of the features underlying normal human preimplantation development which were previously uncharacterized.
Notes
5 To whom correspondence should be addressed at: Department of Reproductive Sciences and Medicine, Division of Obstetrics and Gynaecology, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK. E-mail: deborah.taylor{at}ic.ac.uk 
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Submitted on September 14, 2000; accepted on November 17, 2000.
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